Detailed Description
ICOS
The antibodies according to the invention bind to the extracellular domain of human ICOS. Thus, the antibody binds ICOS-expressing T lymphocytes. Unless the context indicates otherwise, reference herein to "ICOS" or an "ICOS recipient" may be a human ICOS. The sequences of human, cynomolgus and mouse ICOS are shown in the accompanying sequence listing and are available from NCBI as human NCBIID: NP _036224.1, mouse NCBIID: NP _059508.2 and macaque GenBank ID: EHH55098.1 are provided.
Cross reactivity
The antibodies according to the invention are preferably cross-reactive and can, for example, bind the extracellular domain of mouse ICOS as well as human ICOS. The antibody can bind to other non-human ICOS, including ICOS of primates such as cynomolgus monkeys. anti-ICOS antibodies intended for therapeutic use in humans must bind to human ICOS, whereas binding to ICOS of other species would not have direct therapeutic relevance in the human clinical setting. Nevertheless, the data herein indicate that antibodies that bind both human and mouse ICOS have properties that make them particularly useful as agonists and depleting molecules. This may be caused by one or more specific epitopes being targeted by the cross-reactive antibody. However, regardless of the underlying theory, cross-reactive antibodies have high values and are excellent candidates as therapeutic molecules for preclinical and clinical studies.
As explained in the experimental examples, the STIM antibodies described herein use KymouseTMTechnology was generated in which mice have been engineered to lack expression of mouse ICOS (ICOS knockout). ICOS knockout transgenic animals and their use for the production of cross-reactive antibodies are further aspects of the invention.
One method of quantifying the degree of species cross-reactivity of an antibody compared to the antigen of one species is a fold difference in its affinity for the antigen of another species, for example a fold difference in affinity for human ICOS versus mouse ICOS. Affinity can be quantified as KD, which refers to the equilibrium dissociation constant of an antibody-antigen reaction as determined by SPR, where the antibody is in Fab format as described elsewhere herein. Species cross-reactive anti-ICOS antibodies may have a fold difference of 30-fold or less, 25-fold or less, 20-fold or less, 15-fold or less, 10-fold or less, or 5-fold or less in affinity for binding to human and mouse ICOS. In other words, the KD for binding to the extracellular domain of human ICOS may be within 30-fold, 25-fold, 20-fold, 15-fold, 10-fold, or 5-fold of the KD for binding to the extracellular domain of mouse ICOS. An antibody may also be considered to be cross-reactive if the KD for binding to the antigen of both species meets a threshold, for example if the KD for binding to human ICOS and the KD for binding to mouse ICOS are both 10mM or less, preferably 5mM or less, more preferably 1mM or less. The KD can be 10nM or less, 5nM or less, 2nM or less, or 1nM or less. The KD can be 0.9nM or less, 0.8nM or less, 0.7nM or less, 0.6nM or less, 0.5nM or less, 0.4nM or less, 0.3nM or less, 0.2nM or less, or 0.1nM or less.
An alternative measure of cross-reactivity to bind human ICOS and mouse ICOS is the ability of an antibody to neutralize ICOS ligand binding to the ICOS receptor, for example, in an HTRF assay (see example 8). Examples of species cross-reactive antibodies are provided herein, including STIM001, STIM002-B, STIM003, STIM005, and STIM006, each of which was confirmed to neutralize human B7-H2(ICOS ligand) binding to human ICOS and neutralize mouse B7-H2 binding to mouse ICOS in HTRF analysis. When cross-reactive antibodies to human and mouse ICOS are desired, either of these antibodies or variants thereof may be selected. The species cross-reactive anti-ICOS antibody may have an IC50 that inhibits binding of human ICOS to the human ICOS receptor within 25-fold, 20-fold, 15-fold, 10-fold, or 5-fold of the IC50 that inhibits binding of mouse ICOS to the mouse ICOS receptor, as determined in an HTRF assay. An antibody may also be considered to be cross-reactive if both IC50 inhibiting the binding of human ICOS to the human ICOS receptor and IC50 inhibiting the binding of mouse ICOS to the mouse ICOS receptor are 1mM or less, preferably 0.5mM or less, e.g., 30nM or less, 20nM or less, 10nM or less. IC50 may be 5nM or less, 4nM or less, 3nM or less, or 2nM or less. In some cases, IC50 will be at least 0.1nM, at least 0.5nM, or at least 1 nM.
Specificity of
The antibodies according to the invention are preferably specific for ICOS. That is, the antibody binds its epitope on the target protein, ICOS (human ICOS and preferably mouse and/or cynomolgus ICOS as mentioned above), but does not exhibit significant binding to molecules that do not present the epitope, including other molecules in the CD28 gene family. The antibody according to the invention preferably does not bind human CD 28. The antibody also preferably does not bind to mouse or cynomolgus CD 28.
CD28 co-stimulates T cell responses when conjugated to professional antigen presenting cells by their ligands CD80 and CD86 under antigen recognition by TCR. For various in vivo uses of the antibodies described herein, it is considered advantageous to avoid binding to CD 28. The absence of binding of anti-ICOS antibodies to CD28 should allow CD28 to interact with its natural ligand and generate appropriate costimulatory signals for T cell activation. In addition, the absence of binding of anti-ICOS antibodies to CD28 avoids the risk of hyperagonism. Over-stimulation of CD28 can induce the proliferation of resting T cells without the normal need to recognize a cognate antigen via the TCR, potentially leading to uncontrolled activation of T cells and consequent cytokine release syndrome in, inter alia, human subjects. Thus, the non-recognition of CD28 by the antibody according to the invention represents an advantage of the safe clinical use of the antibody in humans.
As discussed elsewhere herein, the invention extends to multispecific antibodies (e.g., bispecific antibodies). A multispecific (e.g., bispecific) antibody may comprise (i) an antibody antigen-binding site for ICOS and (ii) another antigen-binding site that recognizes another antigen (e.g., PD-L1) (optionally an antibody antigen-binding site as described herein). Specific binding of a single antigen binding site can be determined. Thus, antibodies that specifically bind ICOS include antibodies that comprise an antigen binding site that specifically binds ICOS, wherein optionally the antigen binding site for ICOS is comprised within an antigen binding molecule that further comprises one or more additional binding sites for one or more other antigens, such as a bispecific antibody that binds ICOS and PD-L1.
Affinity of
The binding affinity of the antibody to ICOS can be determined. The affinity of an antibody for its antigen can be quantified in terms of the equilibrium dissociation constant KD, the association or association rate (Ka) of antibody-antigen interactions and the ratio of dissociation or dissociation rate (KD). The Kd of antibody-antigen binding, Ka and Kd can be measured using Surface Plasmon Resonance (SPR).
The antibody according to the invention can bind to the EC domain of human ICOS with a KD of 10mM or less, preferably 5mM or less, more preferably 1mM or less. The KD can be 50nM or less, 10nM or less, 5nM or less, 2nM or less, or 1nM or less. The KD can be 0.9nM or less, 0.8nM or less, 0.7nM or less, 0.6nM or less, 0.5nM or less, 0.4nM or less, 0.3nM or less, 0.2nM or less, or 0.1nM or less. The KD may be at least 0.1nM, e.g., at least 0.01nM or at least 0.1 nM.
The quantification of affinity can be performed using SPR with antibodies in Fab format. Suitable methods are as follows:
1. coupling an anti-human (or other species-matched antibody constant region) IgG to a biosensor chip (e.g., a GLM chip), e.g., via primary amine coupling;
2. exposing anti-human IgG (or other matching species antibody) to a test antibody, e.g., in Fab format, to capture the test antibody on the chip;
3. test antigen is passed over the capture surface of the chip at a range of concentrations, for example at 5000nM, 1000nM, 200nM, 40nM, 8nM and 2nM, and at 0nM (i.e. buffer only); and
4. the binding affinity of the test antibody to the test antigen was determined using SPR at 25 ℃. The buffer may be pH7.6, 150mM NaCl, 0.05% detergent (e.g., P20), and 3mM EDTA. The buffer may optionally contain 10mM HEPES. HBS-EP can be used as a working buffer. HBS-EP is available from Teknova, Inc. (California; Cat. No. H8022).
Regeneration of the capture surface can be performed using 10mM glycine pH 1.7. This removes the captured antibody and allows the surface to be used for another interaction. The binding data can be fitted to the 1:1 intrinsic model using standard techniques, such as analyzing the software intrinsic model using ProteOnXPR36 TM.
A variety of SPR instruments are known, e.g. BiacoreTM、ProteOn XPR36TM(biological-) And(Sapidyne Instruments ). A working example of SPR is found in example 7.
As described, affinity can be determined by SPR with antibodies in Fab format, where the antigen is coupled to the chip surface and the test antibody is passed through the chip in Fab format in solution to determine the affinity of the monomeric antibody-antigen interaction. Affinity can be determined at any desired pH (e.g., pH 5.5 or pH 7.6) and at any desired temperature (e.g., 25 ℃ or 37 ℃). As reported in example 7, the antibodies according to the invention bind human ICOS with an apparent affinity of less than 2nM, as determined by SPR using antibodies in monovalent (Fab) format.
Other ways of measuring binding of antibodies to ICOS include Fluorescence Activated Cell Sorting (FACS), e.g., using cells with exogenous surface expression of ICOS (e.g., CHO cells) or activated primary T cells expressing endogenous levels of ICOS. Antibodies that bind to ICOS expressing cells as measured by FACS indicate that the antibodies are capable of binding to the Extracellular (EC) domain of ICOS.
ICOS receptor agonism
ICOS ligand (ICOSL, also known as B7-H2) is a cell surface expression molecule that binds to ICOS receptors [17 ]. This intercellular ligand-receptor interaction contributes to ICOS multimerization on the T cell surface, activation of the receptor, and stimulation of downstream signaling in T cells. In effector T cells, this receptor activation stimulates effector T cell responses.
anti-ICOS antibodies can act as agonists of ICOS, mimicking and even exceeding this stimulatory effect of the natural ICOS ligand on the receptor. Such agonism may result from the ability of the antibody to promote the multimerization of ICOS on T cells. One mechanism for this is where the antibody forms an intercellular bridge between ICOS on the surface of the T cell and a receptor (e.g., Fc receptor) on an adjacent cell (e.g., a B cell, antigen presenting cell, or other immune cell). Another mechanism is where an antibody with multiple (e.g., two) antigen binding sites (e.g., two VH-VL domain pairs) bridges multiple ICOS receptor molecules and thus facilitates multimerization. A combination of these mechanisms may occur.
In such assays, agonism assays may use human ICOS positive T lymphocyte cell lines, such as MJ cells (atccrl-8294), as target T cells for activation, one or more T cell activation measures of the test antibody may be determined and compared to a reference molecule or negative control to determine whether there is a statistically significant (p <0.05) difference in T cell activation achieved by the test antibody as compared to the reference molecule or control, one suitable T cell measure activation is the production of a cytokine, such as IFN γ, TNF α or IL-2.
An agonist antibody may be defined as an antibody that, when tested in a T cell activation assay:
has significantly lower EC50 inducing IFN γ production compared to control antibodies;
induces significantly higher maximal IFN γ production compared to control antibodies;
has significantly lower EC50 inducing IFN γ production compared to ICOSL-Fc;
induces a significantly higher maximum IFN γ production compared to ICOSL-Fc;
has significantly lower EC50 inducing IFN γ production compared to reference antibody C398.4A; and/or
A significantly higher maximum IFN γ production was induced compared to C398.4A.
In vitro T cell assays include the bead binding assay of example 13, the plate binding assay of example 14, and the soluble form assay of example 15.
A significantly lower value or a significantly higher value may for example be at most 0.5 fold difference, at most 0.75 fold difference, at most 2 fold difference, at most 3 fold difference, at most 4 fold difference or at most 5 fold difference compared to a reference value or control value.
Thus, in one example, an antibody according to the invention has significantly lower, e.g., at least 2-fold lower IFN γ -inducing EC50 in an MJ cell activation assay using the antibody in bead-bound format compared to a control.
Bead binding assays use antibodies that bind to the bead surface (and, for control or reference experiments, control antibodies, reference antibodies, or ICOSL-Fc). Magnetic beads can be used, and various species are commercially available, such as DYNABADADS M-450(DYNAL, 5Delaware Drive, Lake Success, N.Y.11042, product Nos. 140.03, 140.04) activated by tosyl. The beads may be coated as described in example 13, or typically by dissolving the coating material in a carbonate buffer (pH 9.6, 0.2M) or other methods known in the art. The use of beads conveniently allows the amount of protein bound to the bead surface to be determined with a good degree of accuracy. Standard Fc protein quantification methods can be used for coupled protein mass on beads. Any suitable method may be used with reference to the correlation criteria within the dynamic range of the analysis. Although DELFIA is exemplified in example 13, ELISA or other methods may be used.
The ability of an antibody to induce expression of IFN γ in such T cells is also measured in vitro primary human T lymphocytes is indicative of ICOS agonism, described herein are two T cell activation assays using primary cells (see example 2): T cell activation assay 1 and T cell activation assay 2 preferably, in T cell activation assay 1 and/or T cell activation assay 2, the antibody will exhibit significant (p <0.05) induction of IFN γ at 5 μ g/ml compared to a control antibody.
Agonism of anti-ICOS antibodies may contribute to their ability to alter the balance of TEff cells in vivo, for example, between TReg and TEff cell populations in a diseased site (e.g., a tumor microenvironment). As discussed elsewhere herein, the ability of an antibody to enhance tumor cell killing by activated ICOS-positive effector T cells can be determined.
T cell dependent killing
Effector T cell function can be determined in a biologically relevant environment using an in vitro co-culture assay in which tumor cells are incubated with relevant immune cells to cause immune cell-dependent killing, wherein the effect of anti-ICOS antibodies on tumor cell killing by TEff is observed.
The ability of the antibodies to enhance tumor cell killing by activated ICOS positive effector T cells can be assayed. anti-ICOS antibodies can stimulate significantly greater (p <0.05) tumor cell killing compared to control antibodies. anti-ICOS antibodies can stimulate similar or greater tumor cell killing in such assays compared to a reference molecule (e.g., ICOS ligand or C398.4 antibody). A similar degree of tumor cell killing can be expressed as an analytical reading of the test antibody that is less than a two-fold difference from the analytical reading of the reference molecule.
ICOS ligand-receptor neutralization potency
The antibody according to the invention may be one which inhibits the binding of ICOS to its ligand ICOSL.
The extent to which an antibody inhibits the binding of an ICOS receptor to its ligand is referred to as its ligand-receptor neutralization potency. Unless otherwise stated, potency is typically expressed as IC50 values in pM. In ligand binding studies, IC50 was the concentration of specific binding levels that reduced receptor binding by a maximum of 50%. IC50 may be calculated by plotting specific receptor binding% as a logarithmic function of antibody concentration and fitting a sigma function (sigmoidal function) to the data using a software program such as prism (graphpad) to generate IC50 values. In HTRF analysis, neutralization efficacy can be determined. A detailed working example of HTRF analysis for ligand-receptor neutralization efficacy is set forth in example 8.
The IC50 value may represent an average of a plurality of measurements. Thus, for example, IC50 values may be obtained from the results of three experiments, and an average IC50 value may then be calculated.
In a ligand-receptor neutralization assay, the IC50 of the antibody can be 1mM or less, e.g., 0.5mM or less. IC50 may be 30nM or less, 20nM or less, 10nM or less, 5nM or less, 4nM or less, 3nM or less, or 2nM or less. IC50 may be at least 0.1nM, at least 0.5nM, or at least 1 nM.
Antibodies
As described in more detail in the examples, we isolated and characterized antibodies of interest designated as: STIM001, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008, and STIM 009. In various aspects of the invention, unless the context indicates otherwise, the antibody may be selected from any of these antibodies, or from a subset of STIM001, STIM002, STIM003, STIM004, and STIM 005. The sequences of each of these antibodies are provided in the accompanying sequence listing, wherein for each antibody the following sequences are shown separately: a nucleotide sequence encoding a VH domain; the amino acid sequence of the VH domain; VH CDR1 amino acid sequence; VH CDR2 amino acid sequence; VH CDR3 amino acid sequence; a nucleotide sequence encoding a VL domain; an amino acid sequence of a VL domain; a VL CDR1 amino acid sequence; a VLCDR2 amino acid sequence; and a VL CDR3 amino acid sequence. The invention encompasses anti-ICOS antibodies having VH and/or VL domain sequences of all antibodies shown in the accompanying sequence tables and/or figures, as well as antibodies comprising HCDR and/or LCDR of those antibodies and optionally having complete heavy chain and/or complete light chain amino acid sequences.
STIM001 has the heavy chain variable region (VH) amino acid of Seq ID No:366Sequences comprising the CDRH1 amino acid sequence of Seq ID No. 363, the CDRH2 amino acid sequence of Seq ID No. 364 and the CDRH3 amino acid sequence of Seq ID No. 365. VHThe heavy chain nucleic acid sequence of the domain is Seq ID No: 367. STIM001 has the light chain variable region (V) of Seq ID No:373L) Amino acid sequence comprising the amino acid sequence CDRL1 of Seq ID No. 370, the amino acid sequence CDRL2 of Seq ID No. 371 and the amino acid sequence CDRL3 of Seq ID No. 372. VLThe light chain nucleic acid sequence of the domain is Seq ID No. 374. VHThe domain may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532, or Seq ID No 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536, and Seq ID No 538. The full-length heavy chain amino acid sequence is Seq ID No. 368 (heavy chain nucleic acid sequence Seq ID No: 369). The full-length light chain amino acid sequence is Seq ID No:375 (light chain nucleic acid sequence Seq ID No: 376).
STIM002 has the heavy chain variable region (V) of Seq ID No:380H) Amino acid sequence comprising the amino acid sequence CDRH1 of Seq ID No:377, the amino acid sequence CDRH2 of Seq ID No:378 and the amino acid sequence CDRH3 of Seq ID No: 379. VHThe heavy chain nucleic acid sequence of the domain is Seq ID No 381. STIM002 has the light chain variable region (V) of Seq ID No:387L) Amino acid sequence comprising the amino acid sequence CDRL1 of Seq ID No. 384, the amino acid sequence CDRL2 of Seq ID No. 385 and the amino acid sequence CDRL3 of Seq ID No. 386. VLThe light chain nucleic acid sequence of the domain is Seq ID No. 388 or Seq ID No. 519. VHThe domains may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No:193, SEQ ID NO,Seq ID No. 195, Seq ID No. 197, Seq ID No. 199, Seq ID No. 201, Seq ID No. 203, Seq ID No. 205, Seq ID No. 340, Seq ID No. 524, Seq ID No. 526, Seq ID No. 528, Seq ID No. 530, Seq ID No. 532, or Seq ID No. 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536, and Seq ID No 538. The full-length heavy chain amino acid sequence is Seq ID No:382 (heavy chain nucleic acid sequence Seq ID No: 383). The full-length light chain amino acid sequence is Seq ID No:389 (light chain nucleic acid sequence Seq ID No:390 or Seq ID No: 520).
STIM002-B has the heavy chain variable region of Seq ID No:394 (V)H) Amino acid sequence comprising the amino acid sequence CDRH1 of Seq ID No:391, the amino acid sequence CDRH2 of Seq ID No:392 and the amino acid sequence CDRH3 of Seq ID No: 393. VHThe heavy chain nucleic acid sequence of the domain is Seq ID No: 395. STIM002-B has the light chain variable region of Seq ID No:401 (V)L) An amino acid sequence comprising the CDRL1 amino acid sequence of Seq ID No. 398, the CDRL2 amino acid sequence of Seq ID No. 399 and the CDRL3 amino acid sequence of Seq ID No. 400. VLThe light chain nucleic acid sequence of the domain is SeqID No. 402. VHThe domain may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532, or Seq ID No 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536 and Seq ID No 536Seq ID No: 538. The full-length heavy chain amino acid sequence is Seq ID No:396 (heavy chain nucleic acid sequence Seq ID No: 397). The full-length light chain amino acid sequence was Seq ID No:403 (light chain nucleic acid sequence SeqID No: 404).
STIM003 heavy chain variable region (V) with Seq ID No:408H) Amino acid sequence comprising the amino acid sequence of CDRH1 of Seq ID No. 405, the amino acid sequence of CDRH2 of Seq ID No. 406 and the amino acid sequence of CDRH3 of Seq ID No. 407. VHThe heavy chain nucleic acid sequence of the domain is Seq ID No 409 or Seq ID No 521. STIM003 light chain variable region (V) having Seq ID No:415L) Amino acid sequence comprising the amino acid sequence CDRL1 of Seq ID No. 412, the amino acid sequence CDRL2 of Seq ID No. 413 and the amino acid sequence CDRL3 of Seq ID No. 414. VLThe light chain nucleic acid sequence of the domain is Seq ID No. 4416. VHThe domain may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532, or Seq ID No 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536, and Seq ID No 538. The full-length heavy chain amino acid sequence is Seq ID No:410 (heavy chain nucleic acid sequence Seq ID No:411 or Seq ID No: 522). The full-length light chain amino acid sequence was Seq ID No:417 (light chain nucleic acid sequence Seq ID No: 418).
STIM004 heavy chain variable region (V) with Seq ID No:422H) Amino acid sequence comprising the amino acid sequence CDRH1 of Seq ID No:419, the amino acid sequence CDRH2 of Seq ID No:420 and the amino acid sequence CDRH3 of Seq ID No: 421. VHThe heavy chain nucleic acid sequence of the domain is Seq ID No: 423. STIM004 light chain variable region (V) with Seq ID No:429L) Amino acid sequenceComprises the amino acid sequence CDRL1 of Seq ID No:426, the amino acid sequence CDRL2 of Seq ID No:427 and the amino acid sequence CDRL3 of Seq ID No: 428. VLThe light chain nucleic acid sequence of the domain is Seq ID No. 430 or Seq ID No. 431. VHThe domain may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532, or Seq ID No 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536, and Seq ID No 538. The full-length heavy chain amino acid sequence is Seq ID No:424 (heavy chain nucleic acid sequence Seq ID No: 425). The full-length light chain amino acid sequence is Seq ID No:432 (light chain nucleic acid sequence Seq ID No:433 or Seq ID No: 434).
STIM005 has the heavy chain variable region (V) of Seq ID No:438H) Amino acid sequence comprising the amino acid sequence CDRH1 of Seq ID No. 435, the amino acid sequence CDRH2 of Seq ID No. 436 and the amino acid sequence CDRH3 of Seq ID No. 437. VHThe heavy chain nucleic acid sequence of the domain is Seq ID No: 439. STIM005 light chain variable region (V) having Seq ID No:445L) Amino acid sequence comprising the amino acid sequence CDRL1 of Seq ID No:442, the amino acid sequence CDRL2 of Seq ID No:443 and the amino acid sequence CDRL3 of Seq ID No: 444. VLThe light chain nucleic acid sequence of the domain is Seq ID No. 446. VHThe domain may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532, or Seq ID No 534. VLDomains can be as described hereinAny combination of light chain constant region sequences of (1), for example, Seq ID No:207, Seq ID No:209, Seq ID No:211, Seq ID No:213, Seq ID No:215, Seq ID No:217, Seq ID No:219, Seq ID No:221, Seq ID No:223, Seq ID No:225, Seq ID No:227, Seq ID No:229, Seq ID No:231, Seq ID No:233, Seq ID No:235, Seq ID No:237, Seq ID No:536, and Seq ID No: 538. The full-length heavy chain amino acid sequence is Seq ID No:440 (heavy chain nucleic acid sequence Seq ID No: 441). The full-length light chain amino acid sequence was Seq ID No:447 (light chain nucleic acid sequence Seq ID No: 448).
STIM006 has the heavy chain variable region (V) of Seq ID No:452H) Amino acid sequence comprising the amino acid sequence CDRH1 of Seq ID No. 449, the amino acid sequence CDRH2 of Seq ID No. 450 and the amino acid sequence CDRH3 of Seq ID No. 451. VHThe heavy chain nucleic acid sequence of the domain is Seq ID No: 453. STIM006 light chain variable region (V) having Seq ID No:459L) Amino acid sequence comprising the amino acid sequence CDRL1 of Seq ID No. 456, the amino acid sequence CDRL2 of Seq ID No. 457 and the amino acid sequence CDRL3 of Seq ID No. 458. VLThe light chain nucleic acid sequence of the domain is Seq ID No: 460. VHThe domain may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532, or Seq ID No 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536, and Seq ID No 538. The full-length heavy chain amino acid sequence is Seq ID No:454 (heavy chain nucleic acid sequence Seq ID No: 455). The full-length light chain amino acid sequence is Seq ID No:461 (light chain nucleic acid sequence Seq ID No: 462).
STIM007 heavy chain with Seq ID No:466Variable region (V)H) An amino acid sequence comprising the amino acid sequence CDRH1 of Seq ID No:463, the amino acid sequence CDRH2 of Seq ID No:464 and the amino acid sequence CDRH3 of Seq ID No: 465. VHThe heavy chain nucleic acid sequence of the domain is Seq ID No. 467. STIM007 light chain variable region (V) with Seq ID No:473L) Amino acid sequence comprising the amino acid sequence CDRL1 of Seq ID No. 470, the amino acid sequence CDRL2 of Seq ID No. 471 and the amino acid sequence CDRL3 of Seq ID No. 472. VLThe light chain nucleic acid sequence of the domain is Seq ID No. 474. VHThe domain may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532, or Seq ID No 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536, and Seq ID No 538. The full-length heavy chain amino acid sequence is Seq ID No:468 (heavy chain nucleic acid sequence Seq ID No: 469). The full-length light chain amino acid sequence was Seq ID No:475 (light chain nucleic acid sequence Seq ID No: 476).
STIM008 heavy chain variable region (V) with Seq ID No:480H) Amino acid sequence comprising the amino acid sequence CDRH1 of Seq ID No:477, the amino acid sequence CDRH2 of Seq ID No:478 and the amino acid sequence CDRH3 of Seq ID No: 479. VHThe heavy chain nucleic acid sequence of the structural domain is Seq ID No: 481. STIM008 light chain variable region (V) with Seq ID No:487L) An amino acid sequence comprising the amino acid sequence CDRL1 of Seq ID No:484, the amino acid sequence CDRL2 of Seq ID No:485 and the amino acid sequence CDRL3 of Seq ID No: 486. VLThe light chain nucleic acid sequence of the domain is Seq ID No. 488. VHThe domains can be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No. 193, Seq ID No. 195, Seq ID No. 197, Seq ID No. 199, Seq ID No. 201, Seq ID No. 203, Seq ID No. 205, Seq ID No. 340, Seq ID No. 524, Seq ID No. 526, Seq ID No. 528, Seq ID No. 530, Seq ID No. 532, or Seq ID No. 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536, and Seq ID No 538. The full-length heavy chain amino acid sequence is Seq ID No. 482 (heavy chain nucleic acid sequence Seq ID No: 483). The full-length light chain amino acid sequence was Seq ID No:489 (light chain nucleic acid sequence Seq ID No: 490).
STIM009 heavy chain variable region (V) with Seq ID No:494H) Amino acid sequence comprising the amino acid sequence CDRH1 of Seq ID No. 491, the amino acid sequence CDRH2 of Seq ID No. 492 and the amino acid sequence CDRH3 of Seq ID No. 493. VHThe heavy chain nucleic acid sequence of the domain is Seq ID No: 495. STIM009 light chain variable region with Seq ID No:501 (V)L) Amino acid sequence comprising the amino acid sequence CDRL1 of Seq ID No:498, the amino acid sequence CDRL2 of Seq ID No:499 and the amino acid sequence CDRL3 of Seq ID No: 500. VLThe light chain nucleic acid sequence of the domain is Seq ID No 502. VHThe domain may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532, or Seq ID No 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536 and Seq ID No 536Seq ID No: 538. The full-length heavy chain amino acid sequence is Seq ID No:496 (heavy chain nucleic acid sequence Seq ID No: 497). The full-length light chain amino acid sequence was Seq ID No:503 (light chain nucleic acid sequence Seq ID No: 504).
Antibodies according to the invention are immunoglobulins or molecules comprising immunoglobulin domains, whether natural or partially or fully synthetically manufactured. The antibody can be an IgG, IgM, IgA, IgD, or IgE molecule or antigen-specific antibody fragment thereof (including but not limited to Fab, F (ab')2, Fv, disulfide-linked Fv, scFv, single domain antibody, closed conformation multispecific antibody, disulfide-linked scFv, diabody), whether derived from any species that naturally produces the antibody or produced by recombinant DNA technology; whether isolated from serum, B cells, hybridomas, transfectomas, yeast or bacteria. Antibodies can be humanized using conventional techniques. The term antibody covers any polypeptide or protein comprising the antigen binding site of an antibody. An antigen binding site (paratope) is the portion of an antibody that binds to and is complementary to an epitope of its target antigen (ICOS).
The term "epitope" refers to a region of an antigen that is bound by an antibody. Epitopes can be defined as structural or functional. Functional epitopes are typically those residues that are a subset of structural epitopes and have an affinity that directly contributes to the interaction. Epitopes can also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, an epitope may include a determinant as a chemically active surface group of a molecule, such as an amino acid, sugar side chain, phosphoryl group, or sulfonyl group, and in certain embodiments, may have specific three-dimensional structural characteristics and/or specific charge characteristics.
An antigen binding site is a polypeptide or domain that comprises one or more CDRs of an antibody and is capable of binding an antigen. For example, the polypeptide comprises a CDR3 (e.g., HCDR 3). For example, the polypeptide comprises the CDRs 1 and 2 (e.g., HCDR1 and HCDR2) or CDRs 1 to CDR3 (e.g., HCDR1 to HCDR3) of the variable domain of the antibody.
The antibody antigen binding site may be provided by one or more antibody variable domains. In an example, the antibody binding site is provided by a single variable domain, for example a heavy chain variable domain (VH domain) or a light chain variable domain (VL domain). In another example, the binding site comprises a VH/VL pair or two or more such pairs. Thus, an antibody antigen-binding site may comprise a VH and a VL.
The antibody may be a whole immunoglobulin, including constant regions, or may be an antibody fragment. An antibody fragment is a portion of an intact antibody, e.g., comprising an antigen binding or variable region of an intact antibody. Examples of antibody fragments include:
(i) fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F (ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region;
(iii) an Fd fragment consisting of the VH and CH1 domains;
(iv) fv fragments consisting of the VL and VH domains of a single arm of an antibody;
(v) dAb fragments (Ward et al, (1989) Nature 341: 544-546; incorporated herein by reference in its entirety) which consist of a VH or VL domain; and
(vi) an isolated Complementarity Determining Region (CDR) that retains specific antigen binding function.
Other examples of antibodies are H2 antibodies comprising dimers of heavy chains (5'-VH- (optional hinge) -CH2-CH3-3') and no light chains.
Single chain antibodies (e.g., ScFv) are commonly used fragments. Multispecific antibodies may be formed from antibody fragments. The antibodies of the invention may be in any such format as desired.
Optionally, the antibody immunoglobulin domain may be fused or conjugated to additional polypeptide sequences and/or labels, tags, toxins or other molecules. The antibody immunoglobulin domain may be fused or conjugated to one or more different antigen binding regions, resulting in a molecule capable of binding a second antigen other than ICOS. The antibodies of the invention may be multispecific antibodies, e.g., bispecific antibodies, comprising (i) an antibody antigen-binding site for ICOS and (ii) another antigen-binding site that recognizes another antigen (e.g., PD-L1) (optionally an antibody antigen-binding site as described herein).
Antibodies typically comprise antibody VH and/or VL domains. Isolated VH and VL domains of antibodies are also part of the invention. Antibody variable domains are portions of the light and heavy chains of antibodies that include the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and Framework Regions (FRs). Thus, within each of the VH and VL domains are CDRs and FRs. The VH domain comprises a set of HCDRs and the VL domain comprises a set of LCDRs. VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain. Each VH and VL is typically composed of three CDRs and four FRs arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. According to the methods used in the present invention, the amino acid positions assigned to the CDRs and FRs can be defined according to Kabat (sequence of the protein of immunological interest (national institute of health, bessel, maryland, 1987 and 1991)) or according to IMGT nomenclature. The antibody may comprise an antibody VH domain comprising VH CDR1, CDR2 and CDR3 and a framework. The antibody may alternatively or additionally comprise an antibody VL domain comprising VL CDRs 1, CDR2 and CDR3 and a framework. Examples of antibody VH and VL domains and CDRs according to the invention are as listed in the accompanying sequence listing which form part of the invention. The CDRs shown in the sequence listing are defined according to the IMGT system [18 ]. All VH and VL sequences, CDR sets, and HCDR and LCDR sets disclosed herein represent aspects and embodiments of the invention. As described herein, a "CDR set" comprises CDR1, CDR2, and CDR 3. Thus, the HCDR groups refer to HCDR1, HCDR2 and HCDR3, and the LCDR groups refer to LCDR1, LCDR2 and LCDR 3. Unless otherwise indicated, "set of CDRs" includes HCDR and LCDR.
The antibodies of the invention may comprise one or more CDRs as described herein, for example CDR3, and optionally also CDR1 and CDR2 to form a CDR set. The CDR or set of CDRs may be a CDR or set of CDRs of any of STIM001, STIM002-B, STIM, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009, or may be variants thereof as described herein.
The invention provides antibodies comprising HCDR1, HCDR2 and/or HCDR3 of any one of antibodies STIM001, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009 and/or LCDR1, LCDR2 and/or LCDR3 (e.g., a set of CDRs) of any of these antibodies. The antibody may comprise a VH CDR set of one of these antibodies. Optionally, the antibody may further comprise a set of VL CDRs of one of these antibodies, and the VL CDRs may be from the same or different antibody as the VH CDRs.
The invention also provides VH domains comprising the disclosed set of HCDRs and/or VL domains comprising the disclosed set of LCDRs.
Typically, the VH domain is paired with the VL domain to provide an antibody antigen binding site, although as discussed further below, either the VH or VL domains alone may be used to bind antigen. The STIM003 VH domain may be paired with the STIM003 VL domain such that an antibody antigen binding site is formed comprising both the STIM003 VH and VL domains. Similar embodiments of other VH and VL domains disclosed herein are provided. In other embodiments, the STIM003 VH is paired with a VL domain other than STIM003 VL. Light chain scrambling is well defined in the art. Likewise, the invention provides similar embodiments of the other VH and VL domains disclosed herein.
Thus, the VH of any one of antibodies STIM001, STIM002, STIM003, STIM004, and STIM005 can be paired with the VL of any one of antibodies STIM001, STIM002, STIM003, STIM004, and STIM 005. In addition, the VH of any one of antibodies STIM001, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008, and STIM009 may be paired with the VL of any one of antibodies STIM001, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008, or STIM 009.
Within an antibody framework, an antibody can comprise one or more CDRs, e.g., a set of CDRs. The framework region may have a sequence of a human germline gene segment. Thus, the antibody may be a human antibody having a VH domain comprising a set of HCDRs in a human germline framework. Typically, the antibody also has a VL domain comprising, for example, the set of LCDRs in the human germline framework. An antibody "gene segment" (e.g., a VH gene segment, a D gene segment, or a JH gene segment) refers to an oligonucleotide having a nucleic acid sequence that is a part of the source of the antibody, e.g., a VH gene segment is an oligonucleotide comprising a nucleic acid sequence from FR1 to CDR3, the part corresponding to a polypeptide VH domain. Human V, D recombines with the J gene segment to produce the VH domain, and human V recombines with the J segment to produce the VL domain. D domain or region refers to the diversity domain or region of an antibody chain. The J domain or region refers to the joining domain or region of an antibody chain. Somatic hypermutation can produce antibody VH or VL domains with framework regions that do not exactly match or align with the corresponding gene segments, but sequence alignment can be used to identify the closest gene segments, and thus the particular combination of gene segments that is the source of a particular VH or VL domain. When aligning an antibody sequence with a gene segment, the antibody amino acid sequence may be aligned with the amino acid sequence encoded by the gene segment, or the antibody nucleotide sequence may be aligned directly with the nucleotide sequence of the gene segment.
Alignments of STIM antibody VH and VL domain sequences against related antibodies and against human germline sequences are shown in fig. 35, 36 and 37.
The antibodies of the invention can be human or chimeric antibodies comprising human variable regions and non-human (e.g., mouse) constant regions. For example, the antibodies of the invention have human variable regions, and optionally also human constant regions.
Thus, the antibody optionally comprises a constant region or portion thereof, e.g., a human antibody constant region or portion thereof. For example, the VL domain may be attached at its C-terminal end to an antibody light chain kappa or lambda constant domain. Similarly, the antibody VH domain may be attached at its C-terminal end to all or part of an immunoglobulin heavy chain constant region (e.g., a CH1 domain or an Fc region) derived from any antibody isotype (e.g., IgG, IgA, IgE, and IgM) and any of the subclasses of isotypes (e.g., IgG1 or IgG 4).
Examples of human heavy chain constant regions are shown in table S1.
The constant region of the antibody of the invention may alternatively be a non-human constant region. For example, when an antibody is produced in a transgenic animal (examples of which are described elsewhere herein), a chimeric antibody comprising a human variable region and a non-human (host animal) constant region can be produced. Some transgenic animals produce fully human antibodies. Other transgenic animals have been engineered to produce antibodies comprising a chimeric heavy chain and a fully human light chain. When the antibody comprises one or more non-human constant regions, these non-human constant regions may be replaced with human constant regions to provide an antibody that is more suitable for administration to a human as a therapeutic composition, since the immunogenicity of the antibody is thereby reduced.
Papain cleavage of antibodies produces two identical antigen binding fragments, also known as "Fab" fragments and "Fc" fragments, that do not have antigen binding activity but have the ability to crystallize. As used herein, "Fab" refers to a fragment of an antibody that includes one constant domain and one variable domain of each of the heavy and light chains. The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. "Fc fragment" refers to the carboxy-terminal portions of two H chains that are linked together by disulfide bonds. The effector functions of antibodies are determined by the sequence of the Fc region, a region also recognized by Fc receptors (fcrs) is found on certain types of cells. The use of pepsin to break down antibodies produces F (ab')2 fragments in which the two arms of the antibody molecule remain linked and contain two antigen binding sites. F (ab')2 fragments have the ability to cross-link antigens.
As used herein, "Fv" refers to the smallest fragment of an antibody that retains both an antigen recognition site and an antigen binding site. This region consists of a dimer of a heavy chain variable domain and a light chain variable domain in close non-covalent or covalent association. In this configuration, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. The six CDRs collectively confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, albeit at a lower affinity than the entire binding site.
The antibodies disclosed herein can be modified to increase or decrease serum half-life. In one example, the following mutants were introduced: one or more of T252L, T254S, or T256F to extend the biological half-life of the antibody. Biological half-life can also be achieved by altering the heavy chain constant region CH1The domain or CL region is extended with a salvage receptor containing a binding epitope taken from CH of the Fc region of IgG2The two loops of the domain, as described in U.S. Pat. nos. 5,869,046 and 6,121,022, the modifications described therein are incorporated herein by reference. In another embodiment, the Fc hinge region of an antibody or antigen binding fragment of the invention is mutated to shorten the biological half-life of the antibody or fragment. Introduction of one or more amino acid mutations into the CH of an Fc hinge fragment2-CH3In the domain interface region, such that the antibody or fragment has reduced staphylococcal protein a (SpA) binding relative to native Fc hinge domain SpA binding. Other methods of extending serum half-life are known to those skilled in the art. Thus, in one embodiment, the antibody or fragment is pegylated. In another embodiment, the antibody or fragment is fused to an albumin binding domain, e.g., an albumin binding single domain antibody (dAb). In another embodiment, the antibody or fragment is PAS (i.e., a genetic fusion of a polypeptide sequence consisting of PAS (XL-Protein GmbH) that forms an uncharged random coil structure with a large hydrodynamic volume). In another embodiment, the antibody or fragment isrPEG (i.e. gene fusion of a non-exact repeat peptide sequence (Amunix, Versartis) with a therapeutic peptide). In another embodiment, the antibody or fragment is ELP-fused (i.e., genetically fused to an ELP repeat (PhaseBio)). These different half-life extending fusions are described in more detail in Strohl, biopharmaceuticals (BioDrugs) (2015)29:215-239, for example the fusions in tables 2 and 6 are incorporated herein by reference.
Antibodies may have modified constant regions that enhance stability. Thus, in one embodiment, the heavy chain constant region comprises a Ser228Pro mutation. In another embodiment, the antibodies and fragments disclosed herein comprise a heavy chain hinge region that has been modified to alter the number of cysteine residues. Such modifications may be used to facilitate the assembly of light and heavy chains or to increase or decrease the stability of an antibody.
Fc effector function: ADCC, ADCP and CDC
As discussed above, anti-ICOS antibodies may be provided in different isotypes and have different constant regions. Examples of human IgG antibody heavy chain constant region sequences are shown in table S1. The Fc region of an antibody is assayed for effector function primarily based on Fc binding, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and antibody-dependent cellular phagocytosis (ADCP) activity. These "cellular effector functions" are involved in recruiting Fc receptor-bearing cells to the site of the target cell, as distinct from effector T cell functions, such that antibody-bound cells are killed. In addition to ADCC and CDC, the ADCP mechanism [19] represents a means of depleting T cells that bind antibodies and thus targeting high ICOS expressing TReg for the deletion.
Cellular effector functions ADCC, ADCP and/or CDC may also be presented by antibodies without an Fc region. An antibody may comprise a plurality of different antigen binding sites, one directed to ICOS and the other directed to a target molecule, wherein engagement of the target molecule induces ADCC, ADCP and/or CDC, e.g. an antibody comprising two scFv regions engaged by a linker, wherein one scFv may engage an effector cell.
An antibody according to the invention may be an antibody which exhibits ADCC, ADCP and/or CDC. Alternatively, an antibody according to the invention may lack ADCC activity, ADCP activity and/or CDC activity. In either case, an antibody according to the invention may comprise, or may optionally lack, an Fc region that binds to one or more types of Fc receptors. The use of different antibody formats and the presence or absence of FcR binding and cellular effector functions makes antibodies suitable for specific therapeutic purposes, as discussed elsewhere herein.
Antibody formats suitable for some therapeutic applications employ wild-type human IgG1 constant regions. The constant region may be an effector-enabling IgG1 constant region, optionally having ADCC activity and/or CDC activity and/or ADCP activity. A suitable wild-type human IgG1 constant region sequence is SEQ ID NO:340(IGHG1 x 01). Other examples of human IgG1 constant regions are shown in table S1.
For testing of candidate therapeutic antibodies in a mouse model of human disease, an effector-positive mouse constant region, such as mouse IgG2a (mIgG2a), may be included instead of an effector-positive human constant region.
The constant region may be engineered for enhanced ADCC and/or CDC and/or ADCP.
The efficacy of Fc-mediated effects can be enhanced by engineering the Fc domain using various established techniques. Such methods increase affinity for certain Fc receptors, thus creating potentially different patterns of enhanced activation. This can be achieved by modifying one or several amino acid residues [20 ]. Human IgG1 constant regions have been shown to enhance binding to Fc receptors, containing specific mutations at residue Asn297 (e.g., N297Q, EU index numbering) or altering glycosylation. Example mutations are one or more of residues selected from 239, 332 and 330 (or equivalent positions in other IgG isotypes) of the human IgG1 constant region. Thus, the antibody may comprise a human IgG1 constant region having one or more mutations independently selected from N297Q, S239D, I332E, and a330L (EU index numbering). Triple mutations (M252Y/S254T/T256E) can be used to enhance binding to FcRn, and other mutations that affect FcRn binding are discussed in table 2 of [21], any of which can be used in the present invention.
Increased affinity for Fc receptors can also be achieved by altering the native glycosylation pattern of the Fc domain, by production, for example, under fucosylation or defucosylation variants [22]. The nonfucosylated antibodies have the trimannosyl core structure of complex N-glycans of Fc without fucose residues. Due to the fact thatEnhanced binding ability, these glycoengineered antibodies that do not have core fucose residues from Fc N-glycans can exhibit more potent ADCC than the fucosylation equivalent. For example, to enhance ADCC, residues in the hinge region may be altered to enhance binding to Fc- γ RIII [23]. Thus, the antibody may comprise a human IgG heavy chain constant region that is a variant of a wild-type human IgG heavy chain constant region, wherein the variant human IgG heavy chain constant region binds human IgG to a greater extent than the wild-type human IgG heavy chain constant regionThe receptors bind with higher affinity to humans selected from the group consisting of FcyRIIB and FcyRIIAA receptor. The antibody can comprise a human IgG heavy chain constant region that is a variant of a wild-type human IgG heavy chain constant region, wherein the variant human IgG heavy chain constant region binds human to a greater extent than the wild-type human IgG heavy chain constant regionHigher affinity binding to humansThe variant human IgG heavy chain constant region can be a variant human IgG1 heavy chain constant region, a variant human IgG2 heavy chain constant region, or a variant human IgG4 heavy chain constant region. In one embodiment, the variant human IgG heavy chain constant region comprises a heavy chain constant region selected from G236D, P238D, S239D, S267E, L328F and L328E (EU index numbering system). In another embodiment, the variant human IgG heavy chain constant region comprises a set of amino acid mutations selected from the group consisting of: S267E and L328F; P238D and L328E; P238D and one or more substitutions selected from the group consisting of: E233D, G237D, H268D, P271G and a 330R; P238D, E233D, G237D, H268D, P271G and a 330R; G236D and S267E; S239D and S267E; V262E, S267E, and L328F; and V264E, S267E, and L328F (EU index numbering system). Enhancement of CDC may be achieved by amino acid changes that enhance affinity for C1q (the first component of a typical complement activation cascade) [24]. Another approach is to generate chimeric Fc domains generated from human IgG1 and human IgG3 segments that exploit the higher affinity of IgG3 for C1q [25]. The antibodies of the invention may comprise amino acids mutated at residues 329, 331 and/or 322 to alter C1q binding and/or to reduce or eliminate CDC activity. In another embodiment, an antibody or antibody fragment disclosed herein may contain an Fc region with modifications at residues 231 and 239, whereby amino acid substitutions are made to alter the ability of the antibody to fix complement. In one embodiment, the antibody or fragment has a constant region comprising one or more mutations selected from the group consisting of E345K, E430G, R344D, and D356R, specifically, a double mutation comprising R344D and D356R (EU index numbering system).
WO2008/137915 describes anti-ICOS antibodies with a modified Fc region having enhanced effector function. The antibodies are reported to mediate enhanced ADCC activity compared to the level of ADCC activity mediated by the parent antibody comprising VH and VK domains and a wild type Fc-region. Antibodies according to the invention may employ such variant Fc regions having effector functions as described therein.
ADCC activity of an antibody can be determined in an assay as described herein. ADCC activity of anti-ICOS antibodies can be determined in vitro using ICOS positive T cell lines as described in example 10. ADCC activity of anti-PD-L1 antibodies can be determined in vitro in an ADCC assay using PD-L1 expressing cells.
For certain applications (e.g. in the case of vaccination) it may be preferred to use antibodies that do not have Fc effector function. Antibodies without a constant region or without an Fc region may be provided-examples of such antibody formats are described elsewhere herein. Alternatively, the antibody may have an effector null constant region. The antibody may have a heavy chain constant region that does not bind to an Fc γ receptor, e.g., the constant region may comprise a Leu235Glu mutation (i.e., wherein the wild-type leucine residue is mutated to a glutamic acid residue). Another optional mutation in the heavy chain constant region is Ser228Pro which increases stability. The heavy chain constant region may be IgG4 comprising both the Leu235Glu mutation and the Ser228Pro mutation. This "IgG 4-PE" heavy chain constant region is effector-null.
The surrogate effector null human constant region is a forbidden IgG 1. The forbidden IgG1 heavy chain constant region may contain alanine at positions 235 and/or 237(EU index numbering), for example, it may be an IgG1 x 01 sequence comprising L235A and/or G237A mutations ("LAGA").
The variant human IgG heavy chain constant region may comprise a reduced IgG to humanHuman beingOr a human beingOne or more amino acid mutations of affinity. In one embodiment of the present invention,expressed on a cell selected from the group consisting of: macrophages, monocytes, B cells, dendritic cells, endothelial cells and activated T cells. In one embodiment, the variant human IgG heavy chain constant region comprises one or more of the following amino acid mutations: G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, a330L, I332E, E333A, K334A, a339T, and P396L (EU index numbering system). At one endIn one embodiment, the variant human IgG heavy chain constant region comprises a set of amino acid mutations selected from the group consisting of: S239D; T256A; K290A; S298A; I332E; E333A; K334A; a 339T; S239D and I332E; S239D, a330L and I332E; S298A, E333A and K334A; G236A, S239D, and I332E; and F243L, R292P, Y300L, V305I and P396L (EU index numbering system). In one embodiment, the variant human IgG heavy chain constant region comprises the S239D, a330L, or I332E amino acid mutation (EU index numbering system). In one embodiment, the variant human IgG heavy chain constant region comprises the S239D and I332E amino acid mutations (EU index numbering system). In one embodiment, the variant human IgG heavy chain constant region is a variant human IgG1 heavy chain constant region comprising amino acid mutations of S239D and I332E (EU index numbering system). In one embodiment, the antibody or fragment comprises an afucosylated Fc region. In another embodiment, the antibody or fragment thereof is defucosylated. In another embodiment, the antibody or fragment is under fucosylation.
An antibody may have a heavy chain constant region that binds to one or more types of Fc receptors but does not induce cellular effector functions, i.e., does not mediate ADCC activity, CDC activity, or ADCP activity. Such constant regions may not be able to bind to a particular Fc receptor responsible for triggering ADCC activity, CDC activity or ADCP activity.
Production and modification of antibodies
Methods for the identification and production of antibodies are well known. Transgenic mice (e.g., Kymouse) can be usedTM、HuMabOr MeMo) A rat (e.g.,) Antibodies are raised against camels, sharks, rabbits, chickens or other non-human animals immunized with ICOS or fragments thereof or synthetic peptides comprising an ICOS sequence motif of interest, followed by optional humanization of the constant and/or variable regions to produce human or humanized antibodies. In examples, display techniques such as yeast display, phage display, or ribosome display may be used, as will be apparent to the skilled person. Standard affinity maturation can be performed in another step after isolation of the antibody leads from transgenic animals, phage display libraries or other libraries, for example using display technology. Representative examples of suitable techniques are described in US20120093818 (Amgen, Inc), which is incorporated herein by reference in its entirety, e.g., in [0309 ]]Segment to [0346 ]]The method set forth in the paragraph.
Immunization of ICOS knockout non-human animals with human ICOS antigen facilitates the generation of antibodies that recognize both human ICOS and non-human ICOS. As described and illustrated in the examples herein, ICOS knockout mice can be immunized with cells expressing human ICOS to stimulate the production of antibodies to human and mouse ICOS in the mice, which can be recovered and tested for binding to human ICOS and mouse ICOS. Antibodies with cross-reactivity can thus be selected, which can be screened for other desired properties as described herein. Methods of producing antibodies to an antigen (e.g., a human antigen) by immunizing an animal with the antigen, wherein expression of the endogenous antigen (e.g., an endogenous mouse antigen) has been knocked out in the animal, can be performed in an animal capable of producing antibodies comprising a human variable domain. The genome of such animals can be engineered to comprise a human or humanized immunoglobulin locus that encodes a human variable region gene segment and optionally an endogenous constant region or a human constant region. Recombination of human variable region gene segments produces human antibodies that may have non-human constant regions or human constant regions. The non-human constant region may then be replaced with a human constant region, wherein the antibody is intended for use in humans. Such methods and knockout transgenic animals are described in WO 2013/061078.
In general, all can be usedAntigen of interest challenge KymouseTM、Or other mice or rats (optionally ICOS knockout mice or rats as mentioned) and lymphocytes (e.g., B cells) are recovered from the antibody-expressing mice. Lymphocytes can be fused with myeloma cell lines to prepare immortal hybridoma cell lines, and such hybridoma cell lines screened and selected to identify hybridoma cell lines that produce antibodies specific for the antigen of interest. DNA encoding the heavy and light chain variable regions may be isolated and ligated to the desired isotype constant regions of the heavy and light chains. Such antibody proteins may be produced in cells (e.g., CHO cells). Alternatively, DNA encoding the antigen-specific chimeric antibody or the light and heavy chain variable domains can be isolated directly from antigen-specific lymphocytes.
First, a high affinity chimeric antibody having a human variable region and a mouse constant region was isolated. Antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, agonism, T cell-dependent killing, neutralizing potency, epitopes, and the like. The mouse constant region is optionally replaced with a desired human constant region to produce a fully human antibody of the invention, such as wild-type or modified IgG1 or IgG4 (e.g., SEQ ID NOs 751, 752, 753 in US2011/0065902 (which is incorporated herein by reference in its entirety). While the constant region selected may vary depending on the particular application, high affinity antigen binding and target-specific features are present in the variable region.
Thus, in another aspect, the invention provides a transgenic non-human mammal having a gene comprising a human or humanized immunoglobulin locus, wherein the mammal does not express ICOS. For example, the mammal may be a knockout mouse or rat, or other experimental animal species. For example KymouseTMThe transgenic mice contain human heavy and light chain immunoglobulin loci inserted at the corresponding endogenous mouse immunoglobulin loci. The transgene according to the inventionThe mammal may be one containing such targeted insertions, or it may contain human heavy and light chain immunoglobulin loci or be randomly inserted into its genome, inserted at loci other than the endogenous Ig loci, or provided with immunoglobulin genes on extra chromosomes or chromosome fragments.
Further aspects of the invention are the use of such non-human mammals for the production of antibodies against ICOS and methods of producing antibodies or antibody heavy chain variable domains and/or light chain variable domains in such mammals.
A method of producing an antibody that binds to the extracellular domain of human and non-human ICOS may comprise: providing a transgenic non-human mammal having a genome comprising a human or humanized immunoglobulin locus, wherein the mammal does not express ICOS, and
(a) immunizing a mammal with a human ICOS antigen (e.g., with cells expressing human ICOS or with a purified recombinant ICOS protein);
(b) isolating antibodies produced by the mammal;
(c) testing the ability of the antibody to bind to human ICOS and non-human ICOS; and
(d) one or more antibodies that bind to human ICOS and non-human ICOS are selected.
Testing for the ability to bind human ICOS and non-human ICOS can be performed using surface plasmon resonance, HTRF, FACS, or any other method described herein. Optionally, binding affinity to human and mouse ICOS is determined. The affinity or fold difference in affinity for binding to human ICOS and mouse ICOS can be determined, and antibodies exhibiting species cross-reactivity can thus be selected (affinity thresholds and fold differences that can be used as selection criteria are exemplified elsewhere herein). The neutralizing potency or the difference in the fold neutralizing potency of an antibody for inhibiting the binding of human and mouse ICOS ligands to human and mouse ICOS receptors may also be determined separately or alternatively, for example in an HTRF assay, according to the manner in which cross-reactive antibodies are screened. Likewise, possible thresholds and fold differences that may be used as selection criteria are illustrated elsewhere herein.
The method may comprise testing the ability of the antibody to bind to non-human ICOS from the same or a different species as the immunized mammal. Thus, when the transgenic mammal is a mouse (e.g., KymouseTM) In time, the antibodies were tested for their ability to bind mouse ICOS. When the transgenic mammal is a rat, the ability of the antibody to bind rat ICOS can be tested. However, it is equally applicable to determining the cross-reactivity of an isolated antibody to non-human ICOS of another species. Thus, antibodies produced in the goat can be tested for binding to rat or mouse ICOS. Optionally, binding to goat ICOS may alternatively or additionally be determined.
In other embodiments, the transgenic non-human mammal can be immunized with non-human ICOS, optionally ICOS of the same mammalian species, but not human ICOS (e.g., an ICOS knockout mouse can be immunized with mouse ICOS). The affinity of the isolated antibody for binding to human ICOS and non-human ICOS was then determined in the same manner, and antibodies that bound both human and non-human ICOS were selected.
Nucleic acids encoding the antibody heavy chain variable domain and/or the antibody light chain variable domain of the selected antibody can be isolated. Such nucleic acids may encode an intact antibody heavy and/or light chain or variable domains without associated constant regions. As mentioned, the encoding nucleotide sequence may be obtained directly from antibody-producing mouse cells, or may be immortalized or fused to B cells to produce hybridomas that express the antibody and encode nucleic acids obtained from such cells. Optionally, the nucleic acid encoding the variable domain is then conjugated to a nucleotide sequence encoding a human heavy chain constant region and/or a human light chain constant region to provide a nucleic acid encoding a human antibody heavy chain and/or a human antibody light chain, e.g., encoding an antibody comprising both a heavy chain and a light chain. As described elsewhere herein, this step is particularly useful when the immunized mammal produces chimeric antibodies with non-human constant regions that are preferably replaced with human constant regions to produce antibodies that will be less immunogenic when administered as a medicament to humans. The provision of a particular human isotype constant region is also important for determining the effector function of an antibody, and a number of suitable heavy chain constant regions are discussed herein.
Other alterations may be made to the nucleic acid encoding the antibody heavy and/or light chain variable domain, for example mutations and variants of the residues as described herein.
The isolated (optionally mutated) nucleic acid may be introduced into a host cell, for example a CHO cell as discussed. The host cell is then cultured under conditions to express the antibody or antibody heavy and/or light chain variable domain in any desired antibody format. Some possible antibody formats are described herein, e.g., intact immunoglobulins, antigen-binding fragments, and other designs.
As discussed, variable domain amino acid sequence variants of either the VH and VL domains or CDRs, the sequences of which are specifically disclosed herein, may be employed according to the present invention.
There are many reasons that it may be desirable to form variants, including optimizing antibody sequences for large scale manufacturing, facilitating purification, enhancing stability or improving suitability for inclusion in a desired pharmaceutical formulation. Protein engineering procedures can be performed at one or more target residues in the antibody sequence, such as substituting an amino acid with a replacement amino acid (optionally, creating a variant containing all naturally occurring amino acids at this position, except possibly Cys and Met), and monitoring the effect on function and expression to determine the optimal substitution. In some cases, it may not be desirable to replace residues with Cys or Met or to introduce these residues into the sequence, for which reason difficulties may arise in manufacturing (e.g. by forming new intramolecular or intermolecular cysteine-cysteine bonds). When a primary candidate has been selected and is being optimized for manufacturing and clinical development, it will often be desirable to alter its antigen binding properties as little as possible or at least retain the affinity and potency of the parent molecule. However, it is also possible to generate variants in order to modulate key antibody characteristics such as affinity, cross-reactivity or neutralisation efficacy.
An antibody can comprise a set of H CDRs and/or L CDRs of any of the disclosed antibodies having one or more amino acid mutations within the set of disclosed H CDRs and/or L CDRs. The mutation may be an amino acid substitution, deletion or insertion. Thus, for example, there may be one or more amino acid substitutions within the disclosed H CDR and/or L CDR sets. For example, up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 mutations, e.g., substitutions, may be present within the H CDR and/or L CDR set. For example, up to 6, 5, 4, 3, or 2 mutations, e.g., substitutions, may be present in HCDR3 and/or up to 6, 5, 4, 3, or 2 mutations, e.g., substitutions, may be present in LCDR 3. The antibody may comprise the HCDR, set of LCDRs or set of 6 (H and L) CDRs shown for any STIM antibody herein, or may comprise a set of CDRs with one or two conservative substitutions.
One or more amino acid mutations may optionally be made in the framework regions of the antibody VH or VL domains disclosed herein. For example, one or more residues that differ from the corresponding human germline fragment sequence may revert to the germline. The sequences of human germline gene segments corresponding to the VH and VL domains of the example anti-ICOS antibodies are indicated in table E12-1, table E12-2 and table E12-3, and the alignment of the antibody VH and VL domains with the corresponding germline sequences is shown in the figure.
The antibody may comprise a VH domain having at least 60%, 70%, 80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity to a VH domain of any of the antibodies shown in the accompanying sequence listing, and/or a VL domain having at least 60%, 70%, 80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity to a VL domain of any of those antibodies. Algorithms that can be used to calculate% identity of two amino acid sequences include, for example, the BLAST, FASTA or Smith-Waterman algorithm (Smith-Waterman algorithm), for example, using default parameters. A particular variant may comprise one or more amino acid sequence alterations (additions, deletions, substitutions and/or insertions of amino acid residues).
Changes may be made in one or more framework regions and/or one or more CDRs. Variants are optionally provided by CDR mutagenesis. The alteration does not typically result in a loss of function, and thus, an antibody comprising the thus altered amino acid sequence may retain the ability to bind ICOS. It may retain the same quantitative binding capacity as an antibody in which no alteration is made, e.g., as measured in an assay as described herein. Antibodies comprising such altered amino acid sequences may have improved ability to bind ICOS.
The alteration may comprise replacing one or more amino acid residues with a non-naturally occurring or non-standard amino acid, modifying one or more amino acid residues to a non-naturally occurring or non-standard form, or inserting one or more non-naturally occurring or non-standard amino acids into the sequence. Examples of the numbering and positions of changes in the sequences of the present invention are described elsewhere herein. Naturally occurring amino acids include the 20 "standard" L-amino acids identified by their standard single letter codes as follows: G. a, V, L, I, M, P, F, W, S, T, N, Q, Y, C, K, R, H, D, E are provided. Non-standard amino acids include any other residue that may be incorporated into the polypeptide backbone or result from modification of an existing amino acid residue. The non-standard amino acids may be naturally occurring or non-naturally occurring.
The term "variant" as used herein refers to a peptide or nucleic acid that differs from a parent polypeptide or nucleic acid by the deletion, substitution, or addition of one or more amino acids or nucleic acids, but retains one or more specific functions or biological activities of the parent molecule. Amino acid substitutions include changes in which an amino acid is substituted for a different naturally occurring amino acid residue. Such substitutions may be categorized as "conservative" substitutions, in which case an amino acid residue contained in a polypeptide is replaced with another naturally occurring amino acid having similar characteristics with respect to polarity, side chain function, or size. Such conservative substitutions are well known in the art. Substitutions encompassed by the present invention may also be "non-conservative" substitutions, wherein an amino acid residue present in a peptide is substituted with an amino acid having different properties, such as a naturally occurring amino acid from a different group (e.g., substitution of a charged or hydrophobic amino acid with alanine), or alternatively, wherein a naturally occurring amino acid is substituted with a non-canonical amino acid. In some embodiments, the amino acid substitution is a conservative substitution. Also encompassed, the term variant when used with reference to a polynucleotide or polypeptide refers to a polynucleotide or polypeptide that differs in primary, secondary, or tertiary structure, respectively, as compared to the reference polynucleotide or polypeptide (e.g., as compared to the wild-type polynucleotide or polypeptide).
In some aspects, we can use "synthetic variants", "recombinant variants", or "chemically modified" polynucleotide variants or polypeptide variants isolated or produced using methods well known in the art. As described below, "modified variants" may include conservative or non-conservative amino acid changes. Polynucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. Some aspects include insertion variants, deletion variants, or substituted variants with amino acid substitutions, including insertions and substitutions of amino acids and other molecules that are not normally present in a peptide sequence on which the variant is based, such as, but not limited to, insertions of ornithine that are not normally present in human proteins. The term "conservative substitution" when describing a polypeptide refers to a change in the amino acid composition of the polypeptide that does not substantially alter the activity of the polypeptide. For example, a conservative substitution refers to a substitution of an amino acid residue for a different amino acid residue with similar chemical properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.). Conservative amino acid substitutions include the substitution of isoleucine or valine for leucine, glutamic for aspartic acids, or serine for threonine. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for one another: 1) alanine (a), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W). (see also Creighton, Proteins, W.H.Freeman and Company (1984), which is incorporated herein by reference in its entirety). In some embodiments, a single substitution, deletion, or addition that alters, adds, or deletes a single amino acid or a small percentage of amino acids may also be considered a "conservative substitution" if the alteration does not decrease the activity of the peptide. Insertions or deletions are typically in the range of about 1 to 5 amino acids. The choice of conservative amino acids may be selected based on the position of the amino acid to be substituted in the peptide, for example where the amino acid is on the outside of the peptide and exposed to a solvent, or on the inside and not exposed to a solvent.
We can select amino acids that will replace existing amino acids based on their position, including their exposure to solvent (i.e., where the amino acid is exposed to solvent or present on the outer surface of the peptide or polypeptide as compared to an internally located amino acid that is not exposed to solvent). The selection of such conservative amino acid substitutions is well known in the art, for example, as described in Dordo et al, journal of molecular biology (J.mol Biol.), 1999,217,721, 739, and Taylor et al, journal of theoretical biology (J.Theor. Biol.) 119 (1986); 205, 218 and s.french and b.robson, journal of molecular evolution (j.mol. evol.) 19(1983) 171. Thus, one can select conservative amino acid substitutions appropriate for amino acids on the exterior of a protein or peptide (i.e., solvent exposed amino acids), for example and without limitation, the following substitutions can be used: y by F, T by S or K, P by A, E by D or Q, N by D or G, R by K, G by N or A, T by S or K, D by N or E, I by L or V, F by Y, S by T or A, R by K, G by N or A, K by R, A by S, K or P.
In alternative embodiments, we can also select conservative amino acid substitutions that are encompassed as applicable to amino acids on the interior of a protein or peptide, e.g., we can use conservative substitutions (i.e., amino acids that are not exposed to solvent) as applicable to amino acids on the interior of a protein or peptide, e.g., but not limited to, we can use the following conservative substitutions: wherein Y is substituted with F, T is substituted with A or S, I is substituted with L or V, W is substituted with Y, M is substituted with L, N is substituted with D, G is substituted with A, T is substituted with A or S, D is substituted with N, I is substituted with L or V, F is substituted with Y or L, S is substituted with A or T, and A is substituted with S, G, T or V. In some embodiments, non-conservative amino acid substitutions are also encompassed within the term variant.
The invention includes a method of producing an antibody comprising a VH and/or VL domain variant of an antibody VH and/or VL domain, said domains being shown in the accompanying sequence listing. Such antibodies can be produced by a method comprising:
(i) providing an antibody VH domain which is an amino acid sequence variant of a parent antibody VH domain by means of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of the parent antibody VH domain,
wherein the parent antibody VH domain is the VH domain of any one of antibodies STIM001, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009 or a VH domain comprising the heavy chain complementarity determining region of any one of those antibodies,
(ii) optionally combining the VH domain thus provided with a VL domain to provide a VH/VL combination, an
(iii) The VH domain or VH/VL domain combinations provided thereby are tested to identify antibodies having one or more desired characteristics.
Desirable characteristics include binding to human ICOS, binding to mouse ICOS, and binding to other non-human ICOS, such as cynomolgus ICOS. Antibodies with comparable or higher affinity for human and/or mouse ICOS can be recognized. Other desirable features include enhancement of effector T cell function indirectly by depletion of immunosuppressive tregs or directly by ICOS signaling activation on T effector cells. Identifying an antibody having a desired characteristic can comprise identifying an antibody having a functional attribute described herein, e.g., its affinity, cross-reactivity, specificity, ICOS receptor agonism, neutralization potency, and/or promoting T cell-dependent killing, any of which can be determined in an assay as described herein.
When included in the method, the VL domain may be that of any one of STIM001, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or may be a variant provided by means of the addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a parent VL domain, wherein the parent VL domain is that of any one of STIM001, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009, or a VL domain comprising the light chain complementarity determining region of any one of those antibodies.
Methods of producing variant antibodies may optionally comprise generating duplicates of the antibody or VH/VL domain combination. The method may further comprise expressing the resulting antibody. It is possible to optionally produce nucleotide sequences corresponding to the VH and/or VL domains of the desired antibody in one or more expression vectors. Suitable expression methods, including recombinant expression in host cells, are set forth in detail herein.
Coding nucleic acids and expression methods
Isolated nucleic acids may be provided, thereby encoding an antibody according to the invention. The nucleic acid may be DNA and/or RNA. Genomic DNA, cDNA, mRNA, or other RNA of synthetic origin, or any combination thereof, can encode the antibody.
The invention provides constructs in the form of plasmids, vectors, transcription or expression cassettes comprising at least one polynucleotide as above. Exemplary nucleotide sequences are included in the sequence listing. Unless the context requires otherwise, reference to a nucleotide sequence as set forth herein encompasses a DNA molecule having the specified sequence, and encompasses an RNA molecule having the specified sequence, wherein U is replaced with T.
The invention also provides recombinant host cells comprising one or more nucleic acids encoding an antibody. Methods of producing the encoded antibodies can comprise expressing the nucleic acids, e.g., by culturing recombinant host cells containing the nucleic acids. Antibodies can be obtained therefrom and can be isolated and/or purified using any suitable technique, then used as appropriate. The method of production may comprise formulating the product into a composition comprising at least one additional component, such as a pharmaceutically acceptable excipient.
Systems for cloning and expressing polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, plant cells, filamentous fungi, yeast and baculovirus systems, and transgenic plants and animals.
Expression of antibodies and antibody fragments in prokaryotic cells is well established in the art. A common bacterial host is escherichia coli (e. As an option for production, expression in eukaryotic cell culture can also be obtained by those skilled in the art. Mammalian cell lines available in the art for expression of heterologous polypeptides include Chinese Hamster Ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, YB2/0 rat myeloma cells, human embryonic kidney cells, human embryonic retina cells, and many others.
The vector may contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other appropriate sequences as desired. Nucleic acids encoding the antibodies can be introduced into host cells. Nucleic acids can be introduced into eukaryotic cells by a variety of methods, including calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses (e.g., vaccinia, or in the case of insect cells, baculovirus). Introduction of nucleic acids into host cells, particularly eukaryotic cells, viral or plasmid based systems may be used. The plasmid system may remain episomal or may be incorporated into the host cell or into an artificial chromosome. Incorporation can be by random or targeted integration of one or more copies at a single or multiple loci. For bacterial cells, suitable techniques include calcium chloride transformation, electroporation, and transfection using phage. The introduction may be followed by expression of the nucleic acid, for example by culturing the host cell under conditions in which the gene is expressed followed by optional isolation or purification of the antibody.
The nucleic acid of the invention may be integrated into the genome (e.g., chromosome) of the host cell. Integration can be facilitated by incorporating sequences that facilitate recombination with the genome according to standard techniques.
The invention also provides methods comprising expressing an antibody in an expression system using the nucleic acids described herein.
Therapeutic uses
The antibodies described herein can be used in methods of treating the human or animal body by therapy. Antibodies have been found to enhance effector T cell responses, which have benefits for a range of diseases or conditions, including the treatment of cancer or solid tumors and in the case of vaccination. Enhanced Teff responses can be achieved using antibodies that modulate the balance or ratio between Teff and tregs that favors Teff activity.
The anti-ICOS antibodies can be used to deplete regulatory T cells and/or enhance effector T cell responses in a patient, and can be administered to a patient to treat a disease or condition amenable to therapy by depleting regulatory T cells and/or enhancing effector T cell responses.
The antibodies of the invention or compositions comprising such antibody molecules or nucleic acids encoding same may be used or provided for use in any such method. Also contemplated is the use of an antibody or a composition comprising the same or a nucleic acid encoding the same for the manufacture of a medicament for use in any such method. The methods generally comprise administering the antibody or composition to a mammal. Suitable formulations and methods of administration are described elsewhere herein.
One contemplated therapeutic use of antibodies is in the treatment of cancer. The cancer may be a solid tumor, such as renal cell carcinoma (renalcell cancer) (optionally renal cell carcinoma such as clear cell renal cell carcinoma), head and neck cancer, melanoma (optionally malignant melanoma), non-small cell lung cancer (e.g., adenocarcinoma), bladder cancer, ovarian cancer, cervical cancer, gastric cancer, liver cancer, pancreatic cancer, breast cancer, testicular germ cell cancer, or metastases of solid tumors such as those listed, or it may be a liquid haematological tumor, such as a lymphoma (e.g., Hodgkin's lymphoma) or a non-Hodgkin's lymphoma, such as diffuse large B-cell lymphoma (DLBCL)) or a leukemia (e.g., acute myeloid leukemia). anti-ICOS antibodies can enhance tumor clearance in melanoma, head and neck cancer, and non-small cell lung cancer with moderate to high mutational loads, as well as other cancers [26 ]. Immunotherapy with anti-ICOS antibodies may offer the prospect of a long lasting cure or long-term remission, even in the case of advanced disease, by enhancing the patient's immune response to their neoplastic lesions.
Although cancer is a distinct disease group, anti-ICOS antibodies offer the possibility of treating a range of different cancers by exploiting the patient's own immune system, with the potential to kill any cancer cell by recognizing mutant or overexpressed epitopes that discriminate cancer cells from normal tissue. By modulating the Teff/Treg balance, anti-ICOS antibodies can achieve and/or promote immune recognition and killing of cancer cells. Although anti-ICOS antibodies are thus therapeutic agents that are useful for a wide variety of cancers, there is a particular classification of cancers in which anti-ICOS therapy is particularly useful, and/or in which anti-ICOS therapy may be effective when other therapeutic agents are ineffective.
One such group is cancers that are positive for ICOS ligand expression. Cancer cells can acquire ICOS ligand expression as has been described for melanoma [27 ]. ICOS ligand expression may provide cells with a selective advantage as surface-expressed ligands bind ICOS on tregs, thereby promoting expansion and activation of tregs and thereby suppressing immune responses against cancer. Survival of cancer cells expressing ICOS ligands may depend on this suppression of the immune system by tregs and will thus be susceptible to treatment with anti-ICOS antibodies targeting tregs. This also applies to cancers derived from cells that naturally express ICOS ligands. Sustained expression of ICOS ligand by these cells also provides survival advantages through immunosuppression. ICOS ligand-expressing cancers may be derived from antigen presenting cells, such as B cells, dendritic cells, and monocytes, and may be liquid hematologic tumors, such as those mentioned herein. Interestingly, these types of cancers have also been shown to be higher in ICOS and FOXP3 expression (TCGA data) — see example 25. Herein, example 20 demonstrates the efficacy of an exemplary anti-ICOS antibody in treating a tumor derived from a cancerous B cell (a20 isogenic cell) expressing an ICOS ligand.
Thus, anti-ICOS antibodies may be used in methods of treating cancers that are positive for ICOS ligand expression. In addition, the cancer to be treated with the anti-ICOS antibody according to the invention may be one that is positive for ICOS and/or FOXP3 expression and optionally also expresses ICOS ligands.
The patient may be tested to determine whether their cancer is positive for expression of the protein of interest (e.g., ICOS ligand, ICOS, and/or FOXP3), for example, by obtaining a test sample (e.g., tumor biopsy) from the patient and determining the expression of the protein of interest. Patients with cancer in which one, two or all of such target protein expression has been characterized as positive are selected for treatment with an anti-ICOS antibody. As discussed elsewhere herein, the anti-ICOS antibody may be used as a monotherapy or in combination with one or more other therapeutic agents.
anti-ICOS antibodies also provide promise for patients whose cancers are refractory to treatment with antibodies or other drugs directed against immune checkpoint molecules such as CTLA-4, PD-1, PD-L1, CD137, GITR, or CD 73. These immunotherapies are effective against some cancers, but in some cases, the cancer may not respond, or it may become unresponsive to continued treatment with the antibody. As with antibodies directed against immune checkpoint inhibitors, anti-ICOS antibodies modulate the immune system of patients-however, anti-ICOS antibodies may succeed when such other antibodies fail. It is shown herein that animals bearing a20B cell lymphoma can be treated with anti-ICOS antibodies to reduce tumor growth, shrink the tumor and actually clear the tumor from the body, whereas therapy with anti-PD-L1 antibody is not superior to controls. The A20 cell line has also been reported to be resistant to CTLA-4 [28 ].
Thus, the anti-ICOS antibody may be used in a method of treating a refractory cancer to treatment with one or more immunotherapies, such as an anti-CTLA-4 antibody, an anti-PD 1 antibody, an anti-PD-L1 antibody, an anti-CD 137 antibody, an anti-GITR antibody, or an anti-CD 73 antibody (any or all). In cases where treatment with an antibody or other drug does not significantly reduce the growth of the cancer, e.g., where the tumor continues to grow or does not decrease in size or where the tumor re-initiates its growth after a response period, the cancer may be characterized as refractory to treatment with the antibody or drug. Non-response to a therapeutic agent can be determined in vitro by testing samples for cancer cell killing or growth inhibition (e.g., tumor biopsy samples), and/or by observing (e.g., using imaging techniques, including MRI) that patients treated with the therapy do not respond to treatment in a clinical setting. Their cancers have been characterized as refractory to treatment with such immunotherapy in patients selected for treatment with anti-ICOS antibodies.
In addition, the anti-ICOS antibodies can be used to treat B cell-derived cancers that are resistant to treatment with the anti-CD 20 antibody. anti-ICOS antibodies represent cancer treatments that fail to respond to, or become resistant to, therapy with an anti-CD 20 antibody, such as rituximab (rituximab). anti-ICOS antibodies may be used as a second line (or additional ) treatment for such cancers. The anti-CD 20 antibody-resistant cancer can be a B cell cancer, such as a B cell lymphoma, such as a diffuse large B cell lymphoma, by testing a sample (e.g., a tumor biopsy sample) for cancer cell killing or growth inhibition with an anti-CD 20 antibody, and/or the resistance of a cancer to anti-CD 20 can be determined ex vivo by observing that a patient treated with an anti-CD 20 antibody does not respond to treatment in a clinical setting. Alternatively or additionally, the cancer (e.g., tumor biopsy samples) may be tested to assess expression of CD20, wherein no or low CD20 expression levels are indicative of loss of sensitivity to the anti-CD 20 antibody.
Samples obtained from the patient can thus be tested to determine the surface expression of target receptors to which a protein of interest, e.g., ICOS ligand, ICOS, FOXP3, and/or another therapeutic agent (e.g., an anti-receptor antibody) is directed. The target receptor may be CD20 (e.g. the receptor to which anti-CD 20 antibody therapy with rituximab is directed) or another receptor, e.g. PD1, EGFR, HER2 or HER 3. Surface expression of ICOS ligand, ICOS, FOXP3 and/or lack or loss of surface expression of the target receptor indicates that the cancer is susceptible to anti-ICOS antibody therapy. An anti-ICOS antibody can be provided for administration to a patient whose cancer is characterized by surface expression of ICOS ligand, ICOS, FOXP3, and/or lack or loss of surface expression of a target receptor, optionally wherein the patient has previously been treated with anti-CTLA 4, anti-PD 1, anti-PD-L1, or with an antibody to a target receptor and has not yet responded to or has ceased responding to treatment with the antibody, as measured, for example, by sustained or regenerative cancer cell growth, e.g., an increase in tumor size.
Any suitable method may be employed to determine whether a cancer cell test is positive for surface expression of a protein such as ICOS ligand, CD20, or other target receptors mentioned herein. A typical method is immunohistochemistry, in which a cell sample (e.g., a tumor biopsy sample) is contacted with an antibody to the target protein, and binding of the antibody is detected using a labeling reagent (typically a second antibody that recognizes the Fc region of the first antibody and carries a detectable label, e.g., a fluorescent label). A positive test sample can be declared, wherein at least 5% of the cells are labeled, as visible by cell staining or other detection markers. Optionally higher cut-off values of e.g. 10% or 25% may be used. The antibody will generally be used in excess. Reagent antibodies to the molecule of interest may be used or may be generated by straightforward methods. For testing for ICOS ligand, antibody MAB1651 is currently available from addi bio (R & D systems) as a mouse IgG that recognizes human ICOS ligands. To test for CD20 expression, rituximab may be used. Detection of mRNA levels for ICOS ligands or target receptors of interest is an alternative technique [27 ].
Another indication that a tumor will respond to treatment with anti-ICOS antibodies is the presence of tregs in the tumor microenvironment. Activated tregs were characterized by ICOS high surface expression and Foxp3 high surface expression. The presence of tregs in particularly an increased number of tumors provides another basis for patients to choose for treatment with anti-ICOS antibodies. Tregs can be detected ex vivo in tumor biopsy samples, for example by immunohistochemistry (analysis of co-expression of Foxp3 with ICOS using antibodies against the target protein, followed by detection of the marker, as described above), or by single cell dispersion of the samples used in FACS using labeled antibodies to ICOS and Foxp 3. FACS methods are exemplified in example 17 and example 18.
The anti-ICOS antibodies are useful for treating cancer associated with an infectious agent, such as a virus-induced cancer. Within this category are head and neck squamous cell carcinomas, cervical carcinomas, Merkel cell carcinomas (Merkel cell carcinoma) and many other cancers. Viruses associated with cancer include HBV, HCV, HPV (cervical, oropharyngeal cancer) and EBV (Burkitt's lymphoma), gastric cancer, Hodgkin's lymphoma, other EBV-positive B-cell lymphomas, nasopharyngeal cancer and post-transplant lymphoproliferative disorder). The International Agency for Research on Cancer (monograph 100B) identified the following major Cancer sites associated with infections:
stomach (Stomach/Gastric): helicobacter pylori (Heliobacter pylori)
Liver: hepatitis B virus, Hepatitis C Virus (HCV), opisthorchia sinensis (Opisthorchia viverrii), Clonorchis sinensis (Clonorchis sinensis)
The cervix: human Papilloma Virus (HPV), with or without HIV
Anal and genital (penis, vulva, vaginal anus): HPV, with or without HIV
Nasopharyngeal: Albstein-Barr Virus (Epstein-Barr Virus; EBV)
Oropharynx: HPV, with or without tobacco or alcohol intake
Kaposi's sarcoma: human herpesvirus type 8, with or without HIV
Non-hodgkin lymphoma: helicobacter pylori, EBV with or without HIV, HCV, human T-lymphotropic virus type 1
Hodgkin's lymphoma: EBV, with or without HIV
Bladder: schistosoma aematobium (Schistosoma haematobium).
The antibodies according to the invention may be used to treat cancers associated with or induced by any of these infectious agents, such as the cancers indicated above.
Stimulation of effector T cell responses may also contribute to immunity against infectious diseases and/or contribute to recovery of infectious diseases in patients. Thus, anti-ICOS antibodies may be used to treat infectious diseases by administering the antibodies to a patient.
Infectious diseases include those caused by pathogens (e.g., bacterial pathogens, fungal pathogens, viral pathogens, or protozoan pathogens), and treatment may promote an immune response in a patient against pathogen infection. An example of a bacterial pathogen is tuberculosis. Examples of viral pathogens are hepatitis B and HIV. An example of a protozoan pathogen is a plasmodium species that causes malaria, such as p.
The antibodies may be used to treat infections, such as those occurring by any of the pathogens mentioned herein. The infection may be a persistent infection or a chronic infection. The infection may be local or systemic. Long-term contact between a pathogen and the immune system can lead to exhaustion or tolerance of the immune system (e.g., as evidenced by increased Treg levels and the Treg: Teff balance favoring the skewing of tregs), and/or to immune evasion by the pathogen through evolution and modification of the displayed pathogen antigens. These characteristics reflect similar processes thought to occur in cancer. anti-ICOS antibodies present a therapeutic approach for treating pathogen infections, such as chronic infections, by modulating the Treg to Teff ratio in favor of Teff and/or other effects described herein.
Patients who have been diagnosed with an infectious disease or infection can be treated. Alternatively, the treatment may be prophylactic and administered to the patient, e.g., as a vaccine, to prevent an infectious disease, as described elsewhere herein.
It has also been suggested that immune responses, particularly IFN γ -dependent systemic immune responses, may be beneficial in the treatment of Alzheimer's disease and other CNS disorders sharing a part of the neuroinflammatory component [29 ]. WO2015/136541 proposes the use of anti-PD-1 antibodies for the treatment of Alzheimer's disease. The anti-ICOS antibodies may be used to treat alzheimer's disease or other neurodegenerative diseases, optionally in combination with one or more other immunomodulatory agents (e.g., antibodies to PD-1).
Combination therapy
Treatment with immunomodulatory antibodies such as anti-CTLA 4, anti-PD 1, or anti-PDL 1, particularly antibodies with Fc effector function, may create a further depleted environment where ICOS highly expressing immunosuppressive cells are beneficial. It may be advantageous to combine anti-ICOS antibodies with such immunomodulators to enhance their therapeutic effect.
Patients who have been treated with immunomodulatory antibodies (e.g., anti-PDL-1, anti-PD-1, anti-CTLA-4) may particularly benefit from treatment with anti-ICOS antibodies. One reason for this is that immunomodulatory antibodies can increase the number of ICOS-positive tregs (e.g., intratumoral tregs) in a patient. This effect has also been observed with certain other therapeutic agents, such as recombinant IL-2. anti-ICOS antibodies can reduce and/or reverse the surge or rise in ICOS + tregs (e.g., intratumoral tregs) caused by treating a patient with another therapeutic agent. Thus, a patient selected for treatment with an anti-ICOS antibody may be a patient who has received treatment with a first therapeutic agent that is an antibody (e.g., an immunomodulatory antibody) or other agent (e.g., IL-2) that increases the number of ICOS + tregs in the patient.
Immunomodulatory agents that may be combined with anti-ICOS antibodies include antibodies to any one of the following: PDL1 (e.g., avilumab), PD-1 (e.g., pembrolizumab or nivolumab), or CTLA-4 (e.g., ipilimumab or tremelimumab). The anti-ICOS antibody may be combined with pidilizumab (pidilizumab). In other embodiments, the anti-ICOS antibody is administered in combination with an anti-CTLA-4 antibody, and/or optionally with a therapeutic antibody that is not an anti-CTLA-4 antibody.
For example, an anti-ICOS antibody may be used in combination with therapy with an anti-PDL 1 antibody. Preferably, the anti-ICOS antibody is an antibody that mediates ADCC, ADCP and/or CDC. Preferably, the anti-PDL 1 antibody is an antibody that mediates ADCC, ADCP and/or CDC. An example of such a combination therapy is the administration of an anti-ICOS antibody with an anti-PDL 1 antibody, where both antibodies have effector positive constant regions. Thus, both the anti-ICOS antibody and the anti-PDL 1 antibody may be able to mediate ADCC, CDC and/or ADCP. Fc effector functions and selection of constant regions are described in detail elsewhere herein, but as an example, anti-ICOS human IgG1 may be combined with anti-PD-L1 human IgG 1. The anti-ICOS antibody and/or the anti-PD-L1 antibody may comprise a wild-type human IgG1 constant region. Alternatively, the effector-positive constant region of an antibody may be one that is engineered to enhance effector function, e.g., enhance CDC, ADCC and/or ADCP. Example antibody constant regions including wild-type human IgG1 sequences and mutations that alter effector function are discussed in detail elsewhere herein.
anti-PDL 1 antibodies that may be combined with anti-ICOS antibodies include:
an anti-PDL 1 antibody, which optionally inhibits binding of PD-1 to PDL1 and/or inhibits PDL1 as an effector positive human IgG 1;
an anti-PD-1 antibody that inhibits the binding of PD-1 to PDL1 and/or PDL 2;
avilumab, a human IgG1 antibody that inhibits binding of PD-1 to PDL-1. See WO 2013/079174;
dolvacizumab (Durvalumab) (or "MEDI 4736"), a variant human IgG1 antibody with mutations L234A, L235A, and 331. See WO 2011/066389;
astuzumab (Atezolizumab), a variant human IgG1 antibody with mutations N297A, D356E and L358M. See US 2010/0203056;
BMS-936559, a human IgG4 antibody comprising the mutation S228P. See WO 2007/005874.
Numerous other examples of anti-PD-L1 antibodies are disclosed herein and other examples are known in the art. Characterization data for many of the anti-PD-L1 antibodies mentioned herein have been published in US9,567,399 and US9,617,338, which are incorporated herein by reference. Example anti-PD-L1 antibodies have VH and/or VL domains comprising HCDR and/or LCDR of any one of: 1D05, 84G09, 1D05HC mutant 1, 1D05HC mutant 2, 1D05HC mutant 3, 1D05HC mutant 4, 1D05LC mutant 1, 1D05LC mutant 2, 1D05LC mutant 3, 411B08, 411C04, 411D07, 385F01, 386H03, 389a03, 413D08, 413G05, 413F09, 414B06 or 416E01 as set forth in US9,567,399 or US9,617,338. The antibody may comprise the VH and VL domains of any of these antibodies, and may optionally comprise a heavy chain and/or a light chain having the heavy chain amino acid sequence and/or the light chain amino acid sequence of any of these antibodies. The VH and VL domains of these anti-PD-L1 antibodies are further described elsewhere herein.
Other example anti-PD-L1 antibodies have VH and/or VL domains of HCDR and/or LCDR comprising KN-035, CA-170, FAZ-053, M7824, ABBV-368, LY-3300054, GNS-1480, yw243.55.s70, REGN3504, or an anti-PD-L1 antibody disclosed in any one of the following: WO2017/034916, WO2017/020291, WO2017/020858, WO2017/020801, WO2016/111645, WO2016/197367, WO2016/061142, WO2016/149201, WO2016/000619, WO2016/160792, WO2016/022630, WO2016/007235, WO2015/179654, WO2015/173267, WO2015/181342, WO2015/109124, WO2015/112805, WO2015/061668, WO2014/159562, WO2014/165082, WO2014/100079, WO2014/055897, WO2013/181634, WO2013/173223, WO2013/079174, WO2012/145493, WO2011/066389, WO2010/077634, WO2010/036959, WO2010/089411 and WO 2010/005874. The antibody may comprise the VH and VL domains of any of these antibodies, and may optionally comprise a heavy chain and/or a light chain having the heavy chain amino acid sequence and/or the light chain amino acid sequence of any of these antibodies. The anti-ICOS antibody used in the combination therapy with anti-PD-L1 may be an antibody of the invention as disclosed herein. Alternatively, the anti-ICOS antibody may comprise the CDRs or VH and/or VL domains of an anti-ICOS antibody disclosed in any one of the following publications:
WO2016154177, US 2016304610-e.g. any of antibodies 7F12, 37a10, 35A9, 36E10, 16G10, 37a10S713, 37a10S714, 37a10S715, 37a10S716, 37a10S717, 37a10S718, 16G10S71, 16G10S72, 16G10S73, 16G10S83, 35A9S79, 35A9S710 or 35A9S 89;
WO16120789, US 2016215059-antibodies, e.g. referred to as 422.2 and/or H2L 5;
WO14033327, EP2892928, US 2015239978-for example referred to as 314-8 and/or antibodies produced by hybridoma CNCM I-4180;
WO12131004, EP2691419, US9376493, US 20160264666-for example the antibody Icos145-1 and/or the antibody produced by the hybridoma CNCM I-4179;
WO 10056804-e.g. antibody JMAb136 or "136";
WO9915553, EP1017723B1, US7259247, US7132099, US7125551, US7306800, US7722872, WO05103086, EP1740617, US8318905, US 8916155-e.g. antibody MIC-944 or 9F 3;
WO983821, US7932358B2, US2002156242, EP0984023, EP1502920, US7030225, US7045615, US7279560, US7226909, US7196175, US7932358, US8389690, WO02070010, EP1286668, EP1374901, US7438905, US 389747405, WO0187981, EP1158004, US6803039, US7166283, US7988965, WO0115732, EP1125585, US7465445, US 7998478-e.g. any JMAb antibody, e.g. JMAb-124, JMAb-126, JMAb-127, JMAb-128, JMAb-135, JMAb-136, JMAb-137, JMAb-138, JMAb-139, JMAb-140, JMAb-141, e.g. any of JMAb-141;
WO 2014/089113-e.g. antibody 17G 9;
WO12174338；
US2016145344；
WO11020024、EP2464661、US2016002336、US2016024211、US8840889；
US8497244。
the anti-ICOS antibody optionally comprises the CDRs of 37a10S713 as disclosed in WO 2016154177. It may comprise the VH and VL domains of 37a10S713, and may optionally have antibody heavy and light chains of 37a10S 713.
The combination of an anti-ICOS antibody with an immunomodulator may provide increased therapeutic effect compared to monotherapy, and may allow therapeutic benefit to be obtained with lower doses of the immunomodulator. Thus, for example, an antibody used in combination with an anti-ICOS antibody (e.g., an anti-PD-L1 antibody, optionally ipilimumab) may be administered at a more common dose of 3mg/kg rather than 10 mg/kg. The dosing regimen of the anti-PD-L1 or other antibody may involve intravenous administration over a 90 minute period every 3 weeks for a total of 4 administrations.
anti-ICOS antibodies can be used to increase the sensitivity of tumors to treatment with anti-PD-L1 antibodies, which can be identified as the dose at which the anti-PD-L1 antibody exerts therapeutic benefit decreases. Thus, an anti-ICOS antibody may be administered to a patient to reduce the dose of anti-PD-L1 antibody effective to treat the patient's cancer or tumor. Administration of the anti-ICOS antibody can reduce the recommended or required dose of anti-PD-L1 antibody administered to the patient to, for example, 75%, 50%, 25%, 20%, 10% or less, as compared to the dose when the anti-PD-L1 antibody is administered without the anti-ICOS. The patient may be treated by administering the anti-ICOS antibody and the anti-PD-L1 antibody in a combination therapy as described herein.
The benefits of combining anti-PD-L1 with anti-ICOS can be extended to a reduction in the dose of each agent when compared to its use as monotherapy. The anti-PD-L1 antibody can be used to reduce the dose at which the anti-ICOS antibody exerts therapeutic benefit, and thus can be administered to a patient to reduce the dose of the anti-ICOS antibody that is effective to treat the patient's cancer or tumor. Thus, the anti-PD-L1 antibody can reduce the recommended or required dose of anti-ICOS antibody administered to a patient to, for example, 75%, 50%, 25%, 20%, 10% or less, as compared to the dose when the anti-ICOS antibody is administered without the anti-PD-L1. The patient may be treated by administering the anti-ICOS antibody and the anti-PD-L1 antibody in a combination therapy as described herein.
As discussed in example 22 herein, treatment with anti-PD-L1 antibody, particularly an antibody with effector-positive Fc, did not show increased expression of ICOS on Teff cells. This is advantageous when such antibodies are administered in combination with an effector-positive anti-ICOS antibody, where an increase in ICOS expression on Teff would undesirably make these cells more susceptible to depletion by the anti-ICOS antibody. In combination with anti-PD-L1, anti-ICOS therapy can thus take advantage of the differential expression of ICOS on Teff versus Treg, preferentially targeting ICOS high Treg for depletion. This in turn mitigates the inhibition of TEff and has the net effect of promoting effector T cell responses in the patient. The effect of targeting immune checkpoint molecules with respect to ICOS expression on T cells has also been studied previously-see figure S6C [30] (supplementary material) in the reference, where treatment with CTLA-4 antibodies and/or anti-PD-1 antibodies is reported to increase the percentage of ICOS-expressing CD4+ tregs. The effect of the therapeutic agent on ICOS expression in tregs and teffs may be a factor in the selection of an appropriate agent for use in combination with the anti-ICOS antibody, it being noted that the effect of the anti-ICOS antibody may be enhanced under conditions where ICOS is highly differentially expressed on tregs and teffs.
As described herein, a single dose of an anti-ICOS antibody (particularly in combination with other therapeutic agents such as an anti-PD-L1 antibody) may be sufficient to provide a therapeutic effect. In tumor therapy, the rationale for this single dose benefit may be that the anti-ICOS antibody mediates its effects at least in part by sufficiently resetting or altering the tumor microenvironment to render the tumor more susceptible to immune attack and/or to the effects of other immunomodulators, such as mentioned. Tumor microenvironment replacement is triggered by, for example, depletion of ICOS-positive tumor-infiltrating T-reg. Thus, for example, a patient may be treated with a single dose of anti-ICOS antibody followed by one or more doses of anti-PD-L1 antibody. The anti-ICOS antibody may be administered in a single dose over a treatment period, e.g., six months or a year, while optionally multiple doses of other agents, e.g., anti-PD-L1 antibody, are administered over the treatment period, preferably at least one such dose administered after treatment with the anti-ICOS antibody.
Other examples of combination therapies include anti-ICOS antibodies in combination with:
-an antagonist of the adenosine A2A receptor ("A2 AR inhibitor");
a CD137 agonist (e.g., an agonist antibody);
antagonists of the enzyme indoleamine-2, 3-dioxygenase ("IDO inhibitors") which catalyze the decomposition of tryptophan. IDO is an immune checkpoint activated in dendritic cells and macrophages that contributes to immune suppression/tolerance.
anti-ICOS antibodies may be used in combination therapy with IL-2 (e.g., recombinant IL-2, e.g., aldesleukin). IL-2 can be administered at High Doses (HD). Typical HD IL-2 therapy involves bolus infusions of over 500,000IU/kg, e.g., 600,000 or 720,000IU/kg, per therapy cycle, where 10 to 15 such bolus infusions are administered every 5 to 10 hour interval, e.g., up to 15 bolus infusions every 8 hours, and the therapy cycle is repeated approximately every 14 to 21 days for up to 6 to 8 cycles. HD IL-2 therapy has been successful in treating tumors, especially melanoma (e.g., metastatic melanoma) and renal cell carcinoma, but its use is limited by the high toxicity of IL-2, which can cause serious side effects.
Treatment with high dose IL-2 has been shown to increase the population of ICOS-positive tregs in cancer patients. [31]. This increase in ICOS + tregs after the first cycle of HD IL-2 therapy is reported to correlate with more severe clinical outcomes-the higher the number of ICOS + tregs, the more severe the prognosis. IL-2 variant F42K has been proposed as a replacement therapy to avoid this undesirable increase in ICOS + Treg cells [32 ]. Yet another approach would be to take advantage of the increase in ICOS + tregs by using the antibodies according to the invention as second line therapeutics.
It may be beneficial to combine IL-2 therapy with an anti-ICOS antibody to exploit the ability of the anti-ICOS antibody to target tregs that are highly expressing ICOS, thereby inhibiting these cells and improving prognosis for patients undergoing IL-2 therapy. Co-administration of IL-2 and an anti-ICOS antibody increases the response rate while avoiding or reducing adverse events in the treated patient population. The combination may permit the use of IL-2 at lower doses compared to IL-2 monotherapy, to reduce the risk or extent of adverse events resulting from IL-2 therapy, while maintaining or enhancing clinical benefit (e.g., reducing tumor growth, eliminating solid tumors, and/or reducing metastasis). In this manner, the addition of anti-ICOS can improve the treatment of patients receiving IL-2, whether High Dose (HD) or Low Dose (LD) IL-2.
Accordingly, one aspect of the invention provides a method of treating a patient by administering to the patient an anti-ICOS antibody, wherein the patient is also treated with IL-2, e.g., HD IL-2. Another aspect of the invention is an anti-ICOS antibody for use in treating a patient, wherein the patient is also treated with IL-2, e.g., HD IL-2. anti-ICOS antibodies are useful as a second line therapy. Thus, the patient may be one who has been treated with IL-2, e.g. has received at least one cycle of HD IL-2 therapy, and has increased ICOS + Treg levels. Cancer cell samples, e.g., tumor biopsy samples, can be analyzed using immunohistochemistry or FACS as described elsewhere herein to detect cells positive for ICOS, Foxp3, ICOSL, and optionally one or more other markers of interest. The method can comprise determining that the patient has increased ICOS + Treg (e.g., in peripheral blood, or in a tumor biopsy) levels after IL-2 treatment, wherein an increased level indicates that the patient will benefit from treatment with the anti-ICOS antibody. The increase in tregs may be associated with control (untreated) individuals or with patients prior to IL-2 therapy. Such patients with higher tregs represent a population that may not benefit from sustained IL-2 treatment alone, but anti-ICOS antibodies in combination with IL-2 therapy or treatment with anti-ICOS antibodies alone provide therapeutic benefit to the patient. Thus, after a positive determination that a patient has increased ICOS + Treg levels, an anti-ICOS antibody and/or additional IL-2 therapy may be administered. Treatment with anti-ICOS antibodies can selectively target and deplete ICOS + tregs relative to other T cell populations in such patients. This provides a therapeutic effect by alleviating the immunosuppression mediated by these cells and thereby enhancing the activity of Teff against target cells, such as tumor cells or infected cells.
Combination therapy with an anti-ICOS antibody and IL-2 can be used for any of the therapeutic indications described herein, and in particular for the treatment of tumors, such as melanoma (e.g., metastatic melanoma) or renal cell carcinoma. Thus, in one example, a patient treated with an anti-ICOS antibody is a patient presenting metastatic melanoma and has been treated with IL-2, e.g., HD IL-2 therapy or LD IL-2 therapy.
In general, when an anti-ICOS antibody is administered to a patient who has received treatment with a first therapeutic agent (e.g., an immunomodulatory antibody) or another agent (e.g., IL-2), the anti-ICOS antibody can be administered after a minimum period of time, e.g., 24 hours, 48 hours, 72 hours, 1 week, or 2 weeks, following administration of the first therapeutic agent. The anti-ICOS antibody may be administered within 2 weeks, 3 weeks, 4 weeks, or 5 weeks after administration of the first therapeutic agent. While it may be desirable to minimize the number of treatments administered to facilitate patient compliance and reduce costs, this does not preclude additional administration of either agent at any time. Indeed, the relevant dosing schedule will be selected to optimize its combined effect, the first therapeutic agent creating an immunological environment in which the effect of anti-ICOS antibodies is particularly beneficial (e.g., higher ICOS + tregs, or antigen release as discussed below). Thus, sequential administration of the first therapeutic agent and subsequent administration of the anti-ICOS antibody may allow the time for the first agent to act to create an in vivo condition in which the anti-ICOS antibody may exhibit its potentiating effect. Different dosing regimens (including simultaneous or sequential combination therapies) are described herein and may be utilized as desired. When the first therapeutic agent is an agent that increases the number of ICOS + tregs in the patient, the treatment regimen for the patient may comprise determining that the patient has an increased number of ICOS + tregs, and then administering an anti-ICOS antibody.
As mentioned, the use of anti-ICOS antibodies in combination therapy may provide the following advantages: reducing the effective dose of the therapeutic agent and/or counteracting the adverse effect of the therapeutic agent increasing ICOS + tregs in the patient. Yet additional therapeutic benefits may be obtained by: a first therapeutic agent is selected such that the antigen is released from the target cell by "immune cell death" and is administered in combination with an anti-ICOS antibody. As mentioned, administration of the anti-ICOS antibody may sequentially follow administration of the first therapeutic agent, followed by administration of the two agents separated by a certain time window as discussed above.
In contrast to apoptosis, immune cell death is a recognized pattern of cell death. It is characterized by the release of ATP and HMGB1 from the cell and the exposure of calreticulin to the plasma membrane [33,34 ].
Immune cell death in the target tissue or target cells causes antigen presenting cells to phagocytose the cells, allowing the display of antigens from the target cells, which in turn induces antigen-specific Teff cells. anti-ICOS antibodies can increase the magnitude and/or duration of the Teff response by acting as agonists of ICOS on Teff cells. In addition, anti-ICOS antibodies can lead to depletion of antigen-specific tregs when the anti-ICOS antibody is an Fc effector function-enabling antibody (e.g., a human IgG1 antibody). Thus, by combining either or both of these effects, modulation of the balance between Teff and Treg cells is beneficial to enhance Teff activity. The combination of anti-ICOS antibodies with a therapy that induces immune cell death in a target tissue or target cell type, e.g., a tumor or cancer cell, thereby promotes the patient's immune response against the target tissue or target cell, thereby assuming a vaccination format in which the vaccine antigen is produced in vivo.
Accordingly, one aspect of the present invention is a method of treating cancer in a patient by vaccinating the cancer cells of the patient in vivo. Another aspect of the invention is an anti-ICOS antibody for use in such methods. The anti-ICOS antibody may be used in a method comprising:
treating the patient with a therapy that causes immune cell death of the cancer cells such that the antigen is presented to antigen-specific effector T cells, and
administering an anti-ICOS antibody to the patient, wherein the anti-ICOS antibody enhances an antigen-specific effector T cell response against the cancer cells.
Therapies that induce immune cell death include radiation (e.g., ionizing the cells with UVC light or gamma rays), chemotherapeutic agents (e.g., oxaliplatin (oxalliplatin), anthracyclines such as doxorubicin (doxorubicin), idarubicin (idarubicin) or mitoxantrone (mitoxantrone), BK channel agonists such as phloretin or pimaric acid, bortezomib (bortezomib), cardiac glycosides (cardiac glycosides), cyclophosphamide, GADD34/PP1 inhibitors with mitomycin, PDT-hypericin, polyinosine-polycytidylic acid, 5-fluorouracil, gemcitabine (gemcitabine), gefitinib (gefitinib), erlotinib (erlotinib) or thapsigargin), and cisplatin antibodies against tumor-associated antigens. The tumor-associated antigen can be any antigen that is overexpressed by tumor cells relative to non-tumor cells (e.g., HER2, CD20, EGFR) of the same tissue. Suitable antibodies include herceptin (herceptin) (anti-HER 2), rituximab (rituximab) (anti-CD 20), or cetuximab (cetuximab) (anti-EGFR).
Thus, it would be advantageous to combine an anti-ICOS antibody with one or more of such treatments. Optionally, an anti-ICOS antibody is administered to a patient who has received such treatment. The anti-ICOS antibody may be administered after a period of time, e.g., 24 hours, 48 hours, 72 hours, 1 week, or 2 weeks, after treatment to induce immune cell death, e.g., between 24 hours and 72 hours after treatment. The anti-ICOS antibody may be administered within 2 weeks, 3 weeks, 4 weeks, or 5 weeks after treatment. Other regimens for combination therapy are discussed elsewhere herein.
Although "in vivo vaccination" has been described above, it is also possible to treat tumour cells to induce immune cell death in vitro, after which the cells may be reintroduced into the patient. Instead of administering an agent or treatment that induces immune cell death directly to the patient, the patient is administered the treated tumor cells. The patient may be treated according to the dosing regimen described above.
As noted, a single dose of anti-ICOS antibody may be sufficient to provide therapeutic benefit. Thus, in the methods of treatment described herein, the anti-ICOS antibody is optionally administered in a single dose. A single dose of anti-ICOS antibody can deplete a patient's tregs with beneficial effects on diseases such as cancer. It has been previously reported that transient ablation of tregs has anti-tumor effects, including slowing tumor progression, treating established tumors and metastases, and prolonging survival, and that ablation can enhance the therapeutic effects of tumor radiation [35 ]. Administration of a single dose of anti-ICOS can provide such depletion of tregs and can be used to enhance the effects of other therapeutic approaches used in combination, such as radiotherapy.
Antibodies against PD-L1
Antibodies to PD-L1 used in combination with anti-ICOS antibodies can comprise the antigen binding site of any anti-PD-L1 antibody, whether as a sole therapeutic agent or in the form of a multispecific antibody as described herein. Numerous examples of anti-PD-L1 antibodies are disclosed herein and other examples are known in the art. Characterization data for many of the anti-PD-L1 antibodies mentioned herein have been published in US9,567,399 and US9,617,338, which are incorporated herein by reference.
1D05 heavy chain variable region having Seq ID No:33 (V)H) Amino acid sequence comprising the amino acid sequence of CDRH1 of Seq ID No. 27(IMGT) or Seq ID No. 30(Kabat), the amino acid sequence of CDRH2 of Seq ID No. 28(IMGT) or Seq ID No. 31(Kabat) and the amino acid sequence of CDRH3 of Seq ID No. 29(IMGT) or Seq ID No. 32 (Kabat). VHThe heavy chain nucleic acid sequence of the domain is Seq ID No: 34. 1D05 light chain variable region having Seq ID No:43(VL) Amino acid sequence comprising the amino acid sequence CDRL1 of Seq ID No:37(IMGT) or Seq ID No:40(Kabat), the amino acid sequence CDRL2 of Seq ID No:38(IMGT) or Seq ID No:41(Kabat) and the amino acid sequence CDRL3 of Seq ID No:39(IMGT) or Seq ID No:42 (Kabat). VLThe light chain nucleic acid sequence of the domain is Seq ID No: 44. VHThe domain may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532, or Seq ID No 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536, and Seq ID No 538. The full-length heavy chain amino acid sequence is Seq ID No. 35 (heavy chain nucleic acid sequence Seq ID No: 36). The full-length light chain amino acid sequence was Seq ID No:45 (light chain nucleic acid sequence Seq ID No: 46).
84G09 heavy chain variable (V) with Seq ID No:13H) A region amino acid sequence comprising the CDRH1 amino acid sequence of Seq ID No:7(IMGT) or Seq ID No:10(Kabat), the CDRH2 amino acid sequence of Seq ID No:8(IMGT) or Seq ID No:11(Kabat), and the CDRH3 amino acid sequence of Seq ID No:9(IMGT) or Seq ID No:12 (Kabat). VHThe heavy chain nucleic acid sequence of the domain is Seq ID No: 14. 84G09 light chain variable region having Seq ID No:23 (V)L) Amino acid sequence comprising the amino acid sequence CDRL1 of Seq ID No:17(IMGT) or Seq ID No:20(Kabat), the amino acid sequence CDRL2 of Seq ID No:18(IMGT) or Seq ID No:21(Kabat) and the amino acid sequence CDRL3 of Seq ID No:19(IMGT) or Seq ID No:22 (Kabat). VLThe light chain nucleic acid sequence of the domain is Seq ID No: 24. VHThe domains may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199. Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532 or Seq ID No 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536, and Seq ID No 538. The full-length heavy chain amino acid sequence is Seq ID No. 15 (heavy chain nucleic acid sequence Seq ID No: 16). The full-length light chain amino acid sequence was Seq ID No:25 (light chain nucleic acid sequence Seq ID No: 26).
1D05HC mutant 1 has the heavy chain variable (V) of Seq ID No:47H) A region amino acid sequence comprising the CDRH1 amino acid sequence of Seq ID No:27(IMGT) or Seq ID No:30(Kabat), the CDRH2 amino acid sequence of Seq ID No:28(IMGT) or Seq ID No:31(Kabat), and the CDRH3 amino acid sequence of Seq ID No:29(IMGT) or Seq ID No:32 (Kabat). 1D05HC mutant 1 having the light chain variable region of Seq ID No:43 (V)L) Amino acid sequence comprising the amino acid sequence of CDRL1 of Seq ID No:37(IMGT) or Seq ID No:40(Kabat), the amino acid sequence of CDRL2 of Seq ID No:38(IMGT) or Seq ID No:41(Kabat), and the amino acid sequence of CDRL3 of Seq ID No:39(IMGT) or Seq ID No:42 (Kabat). VLThe light chain nucleic acid sequence of the domain is Seq ID No. 44. VHThe domain may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532, or Seq ID No 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No. 235, Seq ID No. 237, Seq ID No. 536 and Seq ID No. 538. The full-length light chain amino acid sequence was Seq ID No:45 (light chain nucleic acid sequence Seq ID No: 46).
1D05HC mutant 2 has the heavy chain variable (V) of Seq ID No:48H) A region amino acid sequence comprising the CDRH1 amino acid sequence of Seq ID No:27(IMGT) or Seq ID No:30(Kabat), the CDRH2 amino acid sequence of Seq ID No:28(IMGT) or Seq ID No:31(Kabat), and the CDRH3 amino acid sequence of Seq ID No:29(IMGT) or Seq ID No:32 (Kabat). 1D05HC mutant 2 having the light chain variable region of Seq ID No:43 (V)L) Amino acid sequence comprising the amino acid sequence of CDRL1 of Seq ID No:37(IMGT) or Seq ID No:40(Kabat), the amino acid sequence of CDRL2 of Seq ID No:38(IMGT) or Seq ID No:41(Kabat), and the amino acid sequence of CDRL3 of Seq ID No:39(IMGT) or Seq ID No:42 (Kabat). VLThe light chain nucleic acid sequence of the domain is Seq ID No. 44. VHThe domain may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532, or Seq ID No 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536, and Seq ID No 538. The full-length light chain amino acid sequence was Seq ID No:45 (light chain nucleic acid sequence Seq ID No: 46).
1D05HC mutant 3 has the variable heavy chain of Seq ID No:49 (V)H) A region amino acid sequence comprising the CDRH1 amino acid sequence of Seq ID No:27(IMGT) or Seq ID No:30(Kabat), the CDRH2 amino acid sequence of Seq ID No:28(IMGT) or Seq ID No:31(Kabat), and the CDRH3 amino acid sequence of Seq ID No:29(IMGT) or Seq ID No:32 (Kabat). 1D05HC mutant 3 having the light chain variable region of Seq ID No:43 (V)L) Amino acid sequence, kitContains the CDRL1 amino acid sequence of SEQ ID NO:37(IMGT) or SEQ ID NO:40(Kabat), the CDRL2 amino acid sequence of SEQ ID NO:38(IMGT) or SEQ ID NO:41(Kabat), and the CDRL3 amino acid sequence of SEQ ID NO:39(IMGT) or SEQ ID NO:42 (Kabat). VLThe light chain nucleic acid sequence of the domain is Seq ID No. 44. VHThe domain may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532, or Seq ID No 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536, and Seq ID No 538. The full-length light chain amino acid sequence was Seq ID No:45 (light chain nucleic acid sequence Seq ID No: 46).
1D05HC mutant 4 has the heavy chain variable (V) of Seq ID No:342H) A region amino acid sequence comprising the CDRH1 amino acid sequence of Seq ID No:27(IMGT) or Seq ID No:30(Kabat), the CDRH2 amino acid sequence of Seq ID No:28(IMGT) or Seq ID No:31(Kabat), and the CDRH3 amino acid sequence of Seq ID No:29(IMGT) or Seq ID No:32 (Kabat). 1D05HC mutant 4 has the light chain variable region of Seq ID No:43 (V)L) Amino acid sequence comprising the amino acid sequence of CDRL1 of SEQ ID NO:37(IMGT) or SEQ ID NO:40(Kabat), the amino acid sequence of CDRL2 of SEQ ID NO:38(IMGT) or SEQ ID NO:41(Kabat) and the amino acid sequence of CDRL3 of SEQ ID NO:39(IMGT) or SEQ ID NO:42 (Kabat). VLThe light chain nucleic acid sequence of the domain is Seq ID No. 44. VHThe domains may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532, orSeq ID No:534。VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536, and Seq ID No 538. The full-length light chain amino acid sequence was Seq ID No:45 (light chain nucleic acid sequence Seq ID No: 46).
1D05LC mutant 1 has the heavy chain variable (V) of Seq ID No:33H) A region amino acid sequence comprising the CDRH1 amino acid sequence of Seq ID No:27(IMGT) or Seq ID No:30(Kabat), the CDRH2 amino acid sequence of Seq ID No:28(IMGT) or Seq ID No:31(Kabat), and the CDRH3 amino acid sequence of Seq ID No:29(IMGT) or Seq ID No:32 (Kabat). VHThe heavy chain nucleic acid sequence of the domain is Seq ID No: 34. 1D05LC mutant 1 has the light chain variable region of Seq ID No:50 (V)L) Amino acid sequence comprising the amino acid sequence of CDRL1 of Seq ID No:37(IMGT) or Seq ID No:40(Kabat) and the amino acid sequence of CDRL3 of Seq ID No:39(IMGT) or Seq ID No:42 (Kabat). CDRL2 sequence of 1D05LC mutant 1V from Seq ID No:50 as defined by Kabat or IMGT systemLAnd (4) sequencing. VHThe domain may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532, or Seq ID No 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536, and Seq ID No 538. The full-length heavy chain amino acid sequence is Seq ID No. 35 (heavy chain nucleic acid sequence Seq ID No: 36).
1D05LC mutant 2 has the heavy chain variable (V) of Seq ID No:33H) A region amino acid sequence comprising the CDRH1 amino acid sequence of Seq ID No:27(IMGT) or Seq ID No:30(Kabat), the CDRH2 amino acid sequence of Seq ID No:28(IMGT) or Seq ID No:31(Kabat), and the CDRH3 amino acid sequence of Seq ID No:29(IMGT) or Seq ID No:32 (Kabat). VHThe heavy chain nucleic acid sequence of the domain is Seq ID No: 34. 1D05LC mutant 2 having the light chain variable region of Seq ID No:51 (V)L) Amino acid sequence comprising the amino acid sequence of CDRL1 of SEQ ID NO:37(IMGT) or SEQ ID NO:40(Kabat), the amino acid sequence of CDRL2 of SEQ ID NO:38(IMGT) or SEQ ID NO:41(Kabat) and the amino acid sequence of CDRL3 of SEQ ID NO:39(IMGT) or SEQ ID NO:42 (Kabat). VHThe domain may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532, or Seq ID No 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536, and Seq ID No 538. The full-length heavy chain amino acid sequence is Seq ID No. 35 (heavy chain nucleic acid sequence Seq ID No: 36).
1D05LC mutant 3 has the heavy chain variable (V) of Seq ID No:33H) A region amino acid sequence comprising the CDRH1 amino acid sequence of Seq ID No:27(IMGT) or Seq ID No:30(Kabat), the CDRH2 amino acid sequence of Seq ID No:28(IMGT) or Seq ID No:31(Kabat), and the CDRH3 amino acid sequence of Seq ID No:29(IMGT) or Seq ID No:32 (Kabat). VHThe heavy chain nucleic acid sequence of the domain is Seq ID No: 34. 1D05LC mutant 3 has the light chain variable region of Seq ID No:298 (V)L) Amino acid sequence comprising the amino acid sequence of CDRL1 of Seq ID No:37(IMGT) or Seq ID No:40(Kabat) and the CDRL of Seq ID No:39(IMGT) or Seq ID No:42(Kabat)3 amino acid sequence. CDRL2 sequence of 1D05LC mutant 3V from Seq ID No:298 as defined by the Kabat or IMGT systemLAnd (4) sequencing. VLThe light chain nucleic acid sequence of the domain is Seq ID No. 44. VHThe domain may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532, or Seq ID No 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536, and Seq ID No 538. The full-length heavy chain amino acid sequence is Seq ID No. 35 (heavy chain nucleic acid sequence Seq ID No: 36). The full-length light chain amino acid sequence was Seq ID No:45 (light chain nucleic acid sequence Seq ID No: 46).
411B08 heavy chain variable (V) with Seq ID No:58H) A region amino acid sequence comprising the CDRH1 amino acid sequence of Seq ID No:52(IMGT) or Seq ID No:55(Kabat), the CDRH2 amino acid sequence of Seq ID No:53(IMGT) or Seq ID No:56(Kabat), and the CDRH3 amino acid sequence of Seq ID No:54(IMGT) or Seq ID No:57 (Kabat). VHThe heavy chain nucleic acid sequence of the domain is Seq ID No: 59. 411B08 light chain variable region with Seq ID No:68 (V)L) Amino acid sequence comprising the amino acid sequence CDRL1 of SEQ ID NO:62(IMGT) or SEQ ID No:65(Kabat), the amino acid sequence CDRL2 of SEQ ID No:63(IMGT) or SEQ ID No:66(Kabat) and the amino acid sequence CDRL3 of SEQ ID No:64(IMGT) or SEQ ID No:67 (Kabat). VLThe light chain nucleic acid sequence of the domain is Seq ID No. 69. VHThe domains may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No. 528, Seq ID No. 530, Seq ID No. 532 or Seq ID No. 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536, and Seq ID No 538. The full-length heavy chain amino acid sequence is Seq ID No:60 (heavy chain nucleic acid sequence Seq ID No: 61). The full-length light chain amino acid sequence was Seq ID No:70 (light chain nucleic acid sequence Seq ID No: 71).
411C04 heavy chain variable (V) with Seq ID No:78H) A region amino acid sequence comprising the CDRH1 amino acid sequence of Seq ID No:72(IMGT) or Seq ID No:75(Kabat), the CDRH2 amino acid sequence of Seq ID No:73(IMGT) or Seq ID No:76(Kabat), and the CDRH3 amino acid sequence of Seq ID No:74(IMGT) or Seq ID No:77 (Kabat). VHThe heavy chain nucleic acid sequence of the domain is Seq ID No: 79. 411C04 light chain variable region with Seq ID No:88 (V)L) Amino acid sequence comprising the amino acid sequence CDRL1 of SEQ ID NO:82(IMGT) or SEQ ID NO:85(Kabat), the amino acid sequence CDRL2 of SEQ ID NO:83(IMGT) or SEQ ID NO:86(Kabat) and the amino acid sequence CDRL3 of SEQ ID NO:84(IMGT) or SEQ ID NO:87 (Kabat). VLThe light chain nucleic acid sequence of the domain is Seq ID No. 89. VHThe domain may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532, or Seq ID No 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536, and Seq ID No 538. Full lengthThe heavy chain amino acid sequence is Seq ID No:80 (heavy chain nucleic acid sequence Seq ID No: 81). The full-length light chain amino acid sequence was Seq ID No:90 (light chain nucleic acid sequence Seq ID No: 91).
411D07 heavy chain variable (V) with Seq ID No:98H) A region amino acid sequence comprising the CDRH1 amino acid sequence of Seq ID No:92(IMGT) or Seq ID No:95(Kabat), the CDRH2 amino acid sequence of Seq ID No:93(IMGT) or Seq ID No:96(Kabat), and the CDRH3 amino acid sequence of Seq ID No:94(IMGT) or Seq ID No:97 (Kabat). VHThe heavy chain nucleic acid sequence of the structural domain is Seq ID No. 99. 411D07 light chain variable region with Seq ID No:108 (V)L) Amino acid sequence comprising the amino acid sequence CDRL1 of SEQ ID NO:102(IMGT) or SEQ ID NO:105(Kabat), the amino acid sequence CDRL2 of SEQ ID NO:103(IMGT) or SEQ ID NO:106(Kabat) and the amino acid sequence CDRL3 of SEQ ID NO:104(IMGT) or SEQ ID NO:107 (Kabat). VLThe light chain nucleic acid sequence of the domain is Seq ID No. 109. VHThe domain may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532, or Seq ID No 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536, and Seq ID No 538. The full-length heavy chain amino acid sequence is Seq ID No:100 (heavy chain nucleic acid sequence Seq ID No: 101). The full-length light chain amino acid sequence was Seq ID No:110 (light chain nucleic acid sequence Seq ID No: 111).
385F01 heavy chain variable (V) with Seq ID No:118H) A region amino acid sequence comprising the CDRH1 amino acid sequence of Seq ID No:112(IMGT) or Seq ID No:115(Kabat), the CDRH2 amino acid sequence of Seq ID No:113(IMGT) or Seq ID No:116(Kabat), and Seq ID No:114 (see above)IMGT), or the CDRH3 amino acid sequence of Seq ID No. 117 (Kabat). VHThe heavy chain nucleic acid sequence of the domain is Seq ID No: 119. 385F01 light chain variable region having Seq ID No:128 (V)L) Amino acid sequence comprising the amino acid sequence CDRL1 of SEQ ID NO:122(IMGT) or SEQ ID NO:125(Kabat), the amino acid sequence CDRL2 of SEQ ID NO:123(IMGT) or SEQ ID NO:126(Kabat) and the amino acid sequence CDRL3 of SEQ ID NO:124(IMGT) or SEQ ID NO:127 (Kabat). VLThe light chain nucleic acid sequence of the domain is Seq ID No. 129. VHThe domain may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532, or Seq ID No 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536, and Seq ID No 538. The full-length heavy chain amino acid sequence is Seq ID No:120 (heavy chain nucleic acid sequence Seq ID No: 121). The full-length light chain amino acid sequence was Seq ID No:130 (light chain nucleic acid sequence SeqID No: 131).
386H03 variable heavy chain (V) with Seq ID No:158H) A region amino acid sequence comprising the CDRH1 amino acid sequence of Seq ID No:152(IMGT) or Seq ID No:155(Kabat), the CDRH2 amino acid sequence of Seq ID No:153(IMGT) or Seq ID No:156(Kabat), and the CDRH3 amino acid sequence of Seq ID No:154(IMGT) or Seq ID No:157 (Kabat). VHThe heavy chain nucleic acid sequence of the domain is Seq ID No. 159. 386H03 light chain variable region with Seq ID No:168 (V)L) Amino acid sequence comprising the amino acid sequence of CDRL1 of SEQ ID NO:162(IMGT) or SEQ ID NO:165(Kabat), the amino acid sequence of CDRL2 of SEQ ID NO:163(IMGT) or SEQ ID NO:166(Kabat) and the amino acid sequence of CDRL3 of SEQ ID NO:164(IMGT) or SEQ ID NO:167 (Kabat). VLLight chain of structural domainThe nucleic acid sequence is Seq ID No. 169. VHThe domain may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532, or Seq ID No 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536, and Seq ID No 538. The full-length heavy chain amino acid sequence is Seq ID No:160 (heavy chain nucleic acid sequence Seq ID No: 161). The full-length light chain amino acid sequence was Seq ID No:170 (light chain nucleic acid sequence SeqID No: 171).
389A03 heavy chain variable (V) with Seq ID No:178H) A region amino acid sequence comprising the CDRH1 amino acid sequence of Seq ID No:172(IMGT) or Seq ID No:175(Kabat), the CDRH2 amino acid sequence of Seq ID No:173(IMGT) or Seq ID No:176(Kabat), and the CDRH3 amino acid sequence of Seq ID No:174(IMGT) or Seq ID No:177 (Kabat). VHThe heavy chain nucleic acid sequence of the domain is Seq ID No. 179. 389A03 light chain variable region with Seq ID No:188 (V)L) Amino acid sequence comprising the amino acid sequence CDRL1 of SEQ ID NO:182(IMGT) or SEQ ID NO:185(Kabat), the amino acid sequence CDRL2 of SEQ ID NO:183(IMGT) or SEQ ID NO:186(Kabat) and the amino acid sequence CDRL3 of SEQ ID NO:184(IMGT) or SEQ ID NO:187 (Kabat). VLThe light chain nucleic acid sequence of the domain is Seq ID No. 189. VHThe domain may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532, or Seq ID No 534. VLThe domain may be associated with any of the light chain constant region sequences described hereinOne combination, for example, Seq ID No. 207, Seq ID No. 209, Seq ID No. 211, Seq ID No. 213, Seq ID No. 215, Seq ID No. 217, Seq ID No. 219, Seq ID No. 221, Seq ID No. 223, Seq ID No. 225, Seq ID No. 227, Seq ID No. 229, Seq ID No. 231, Seq ID No. 233, Seq ID No. 235, Seq ID No. 237, Seq ID No. 536, and Seq ID No. 538. The full-length heavy chain amino acid sequence is Seq ID No:180 (heavy chain nucleic acid sequence Seq ID No: 181). The full-length light chain amino acid sequence is Seq ID No:190 (light chain nucleic acid sequence SeqID No: 191).
413D08 heavy chain variable (V) with Seq ID No:138H) A region amino acid sequence comprising the CDRH1 amino acid sequence of Seq ID No:132(IMGT) or Seq ID No:135(Kabat), the CDRH2 amino acid sequence of Seq ID No:133(IMGT) or Seq ID No:136(Kabat), and the CDRH3 amino acid sequence of Seq ID No:134(IMGT) or Seq ID No:137 (Kabat). VHThe heavy chain nucleic acid sequence of the domain is Seq ID No. 139. 413D08 light chain variable region having Seq ID No:148 (V)L) Amino acid sequence comprising the amino acid sequence of CDRL1 of SEQ ID NO:142(IMGT) or SEQ ID NO:145(Kabat), the amino acid sequence of CDRL2 of SEQ ID NO:143(IMGT) or SEQ ID NO:146(Kabat) and the amino acid sequence of CDRL3 of SEQ ID NO:144(IMGT) or SEQ ID NO:147 (Kabat). VLThe light chain nucleic acid sequence of the domain is Seq ID No: 149. VHThe domain may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532, or Seq ID No 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536, and Seq ID No 538. The full-length heavy chain amino acid sequence is Seq ID No:140 (heavy chain nucleic acid sequence Seq ID No: 141). The full-length light chain amino acid sequence is Seq ID No:150(light chain nucleic acid sequence SeqID No: 151).
413G05 heavy chain variable (V) with Seq ID No:244H) A region amino acid sequence comprising the CDRH1 amino acid sequence of Seq ID No:238(IMGT) or Seq ID No:241(Kabat), the CDRH2 amino acid sequence of Seq ID No:239(IMGT) or Seq ID No:242(Kabat), and the CDRH3 amino acid sequence of Seq ID No:240(IMGT) or Seq ID No:243 (Kabat). VHThe heavy chain nucleic acid sequence of the domain is Seq ID No. 245. 413G05 light chain variable region having Seq ID No:254 (V)L) Amino acid sequence comprising the amino acid sequence CDRL1 of SEQ ID NO:248(IMGT) or SEQ ID No:251(Kabat), the amino acid sequence CDRL2 of SEQ ID No:249(IMGT) or SEQ ID No:252(Kabat) and the amino acid sequence CDRL3 of SEQ ID No:250(IMGT) or SEQ ID No:253 (Kabat). VLThe light chain nucleic acid sequence of the domain is Seq ID No. 255. VHThe domain may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532, or Seq ID No 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536, and Seq ID No 538. The full-length heavy chain amino acid sequence is Seq ID No:246 (heavy chain nucleic acid sequence Seq ID No: 247). The full-length light chain amino acid sequence was Seq ID No:256 (light chain nucleic acid sequence SeqID No: 257).
413F09 heavy chain variable (V) with Seq ID No:264H) A region amino acid sequence comprising the CDRH1 amino acid sequence of Seq ID No:258(IMGT) or Seq ID No:261(Kabat), the CDRH2 amino acid sequence of Seq ID No:259(IMGT) or Seq ID No:262(Kabat), and the CDRH3 amino acid sequence of Seq ID No:260(IMGT) or Seq ID No:263 (Kabat). VHThe heavy chain nucleic acid sequence of the domain is Seq IDNo. 265. 413F09 light chain variable region having Seq ID No:274 (V)L) Amino acid sequence comprising the amino acid sequence CDRL1 of SEQ ID NO:268(IMGT) or SEQ ID NO:271(Kabat), the amino acid sequence CDRL2 of SEQ ID NO:269(IMGT) or SEQ ID NO:272(Kabat) and the amino acid sequence CDRL3 of SEQ ID NO:270(IMGT) or SEQ ID NO:273 (Kabat). VLThe light chain nucleic acid sequence of the domain is Seq ID No: 275. VHThe domain may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532, or Seq ID No 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536, and Seq ID No 538. The full-length heavy chain amino acid sequence is Seq ID No:266 (heavy chain nucleic acid sequence Seq ID No: 267). The full-length light chain amino acid sequence was Seq ID No:276 (light chain nucleic acid sequence SeqID No: 277).
414B06 heavy chain variable (V) with Seq ID No:284H) A region amino acid sequence comprising the CDRH1 amino acid sequence of Seq ID No:278(IMGT) or Seq ID No:281(Kabat), the CDRH2 amino acid sequence of Seq ID No:279(IMGT) or Seq ID No:282(Kabat), and the CDRH3 amino acid sequence of Seq ID No:280(IMGT) or Seq ID No:283 (Kabat). VHThe heavy chain nucleic acid sequence of the domain is Seq ID No: 285. 414B06 light chain variable region having Seq ID No:294 (V)L) Amino acid sequence comprising the amino acid sequence of CDRL1 of SEQ ID NO:288(IMGT) or SEQ ID NO:291(Kabat), the amino acid sequence of CDRL2 of SEQ ID NO:289(IMGT) or SEQ ID NO:292(Kabat), and the amino acid sequence of CDRL3 of SEQ ID NO:290(IMGT) or SEQ ID NO:293 (Kabat). VLThe light chain nucleic acid sequence of the domain is Seq ID No: 295. VHThe domains may be in the heavy chain constant region sequences described hereinAny combination of (1) and (2), for example, Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532, or Seq ID No: 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No 217, Seq ID No 219, Seq ID No 221, Seq ID No 223, Seq ID No 225, Seq ID No 227, Seq ID No 229, Seq ID No 231, Seq ID No 233, Seq ID No 235, Seq ID No 237, Seq ID No 536, and Seq ID No 538. The full-length heavy chain amino acid sequence is Seq ID No. 286 (heavy chain nucleic acid sequence Seq ID No: 287). The full-length light chain amino acid sequence was Seq ID No:296 (light chain nucleic acid sequence SeqID No: 297).
416E01 heavy chain variable region with Seq ID No:349 (V)H) Amino acid sequence comprising the amino acid sequence of CDRH1 of Seq ID No:343(IMGT) or Seq ID No:346(Kabat), the amino acid sequence of CDRH2 of Seq ID No:344(IMGT) or Seq ID No:347(Kabat) and the amino acid sequence of CDRH3 of Seq ID No:345(IMGT) or Seq ID No:348 (Kabat). VHThe heavy chain nucleic acid sequence of the domain is Seq ID No: 350. 416E01 light chain variable region (V) with Seq ID No:359L) Amino acid sequence comprising the amino acid sequence of CDRL1 of SEQ ID NO:353(IMGT) or SEQ ID NO:356(Kabat), the amino acid sequence of CDRL2 of SEQ ID NO:354(IMGT) or SEQ ID NO:357(Kabat), and the amino acid sequence of CDRL3 of SEQ ID NO:355(IMGT) or SEQ ID NO:358 (Kabat). VLThe light chain nucleic acid sequence of the domain is Seq ID No: 360. VHThe domain may be combined with any of the heavy chain constant region sequences described herein, e.g., Seq ID No 193, Seq ID No 195, Seq ID No 197, Seq ID No 199, Seq ID No 201, Seq ID No 203, Seq ID No 205, Seq ID No 340, Seq ID No 524, Seq ID No 526, Seq ID No 528, Seq ID No 530, Seq ID No 532, or Seq ID No 534. VLThe domains may be combined with any of the light chain constant region sequences described herein, e.g., Seq ID No 207, Seq ID No 209, Seq ID No 211, Seq ID No 213, Seq ID No 215, Seq ID No. 217, Seq ID No. 219, Seq ID No. 221, Seq ID No. 223, Seq ID No. 225, Seq ID No. 227, Seq ID No. 229, Seq ID No. 231, Seq ID No. 233, Seq ID No. 235, Seq ID No. 237, Seq ID No. 536, and Seq ID No. 538. The full-length heavy chain amino acid sequence is Seq ID No:351 (heavy chain nucleic acid sequence Seq ID No: 352). The full-length light chain amino acid sequence was Seq ID No:361 (light chain nucleic acid sequence SeqID No: 362).
Antibody-drug conjugates
anti-ICOS antibodies can be used as carriers for cytotoxic agents to target tregs. As reported in example 18, tregs localized in the Tumor Microenvironment (TME) strongly express ICOS. ICOS is more strongly expressed on intratumoral tregs than on intratumoral Teff or peripheral tregs. Thus, anti-ICOS antibodies labeled with toxic drugs or prodrugs can preferentially target tregs in TME to deliver toxic payloads, thereby selectively inhibiting those cells. Such targeting of cytotoxic agents provides an additional way to remove the immunosuppressive effects of tregs, thereby altering the Treg to Teff balance in favor of Teff activity, and may be used as an alternative to or in combination with any one or more of the other therapeutic approaches discussed herein (e.g., Fc effector-mediated inhibition of tregs, agonism of effector T cells).
Accordingly, the present invention provides an anti-ICOS antibody conjugated to a cytotoxic drug or prodrug. In the case of a prodrug, the prodrug can be activated in the TME or other target site of therapeutic activity to produce a cytotoxic agent. Activation may be in response to, for example, a light-activated trigger, such as the use of near-infrared light to activate the light absorber conjugate [36 ]. The spatially selective activation of the prodrug further enhances the cytotoxic effects of the antibody-drug conjugate, thereby combining with the high ICOS expression on intratumoral tregs to provide a highly selective cytotoxic effect against these cells.
For use in antibody-drug conjugates, the cytotoxic drug or prodrug is preferably non-immunogenic and non-toxic (dormant or inactive) during the time the antibody-drug conjugate is circulating in the blood. Preferably, the cytotoxic drug (or prodrug when activated) is effective-for example, two to four molecules of the drug may be sufficient to kill the target cell. The light-activated prodrug is a silicon phthalocyanine dye (IRDye 700DX) that induces lethal damage to the cell membrane after near-infrared exposure. Cytotoxic drugs include antimitotic agents such as monomethyl auristatin e (monomethylauristatin e) and microtubule inhibitors such as maytansine derivatives (maytansine derivatives), e.g., maytansine (mertansine), DM1, entecasin (emtansine).
Conjugation of the drug (or prodrug) to the antibody will typically be via a linker. The linker may be a cleavable linker, such as a disulfide bond, hydrazone, or peptide bond. A cathepsin-cleavable linker may be used, allowing the drug to be released by the cathepsin in the tumour cells. Alternatively, non-cleavable linkers, such as thioether linkages, may be used. Additional linkers and/or spacers may also be included.
The antibody in the antibody-drug conjugate can be an antibody fragment, such as Fab'2 or other antigen binding fragments as described herein, because the small size of such fragments can aid in penetration to a tissue site (e.g., a solid tumor).
The anti-ICOS antibody according to the present invention may be provided as an immunocytokine. anti-ICOS antibodies may also be administered with immunocytokines in combination therapy. Various examples of antibodies for use with anti-ICOS in combination therapy are described herein, and any of these antibodies (e.g., anti-PD-L1 antibody) can be provided as an immunocytokine for use in the present invention. Immunocytokines include antibody molecules conjugated to cytokines such as IL-2. anti-ICOS: IL-2 conjugates and anti-PD-L1: IL-2 conjugates are thus further aspects of the invention.
IL-2 cytokine can be highAffinity IL-2 receptor and/or intermediate affinity (αβ) IL-2 receptor activity IL-2 as used in immunocytokines can be human wild-typeA type IL-2 or a variant IL-2 cytokine with one or more amino acid deletions, substitutions or additions, e.g. IL-2 with 1 to 10 amino acid deletions at the N-terminus. Other IL-2 variants include mutations R38A or R38Q.
An example anti-PD-L1 immunocytokine comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the heavy chain comprises in an N-terminal to C-terminal direction:
a)VHa domain comprising CDRH1, CDRH2, and CDRH 3; and
b) a heavy chain constant region;
and wherein the light chain comprises in the N-terminal to C-terminal direction:
c)VLa domain comprising CDRL1, CDRL2, and CDRL 3;
d) light chain constant region (C)L)；
e) Optionally, a linker (L); and
f) an IL-2 cytokine;
wherein VHDomains and VLThe domain consists of an antigen binding site that specifically binds to human PD-L1; and is
Wherein the immunocytokine comprises VHA domain comprising a motif X1GSGX2YGX3X4CDRH3 of FD (SeqID No:609), where X1、X2And X3Independently any amino acid, and X4Present or absent, and if present, may be any amino acid.
The VH and VL domains may be those of any of the anti-PD-L1 antibodies mentioned herein, for example the 1D05VH and VL domains.
The IL-2 may be human wild-type IL-2 or a variant IL-2.
Vaccination
The anti-ICOS antibody may be provided in a vaccine composition or co-administered with a vaccine formulation. ICOS is associated with T-follicular helper cell formation and germinal center response [37 ]. Agonist ICOS antibodies thus have potential clinical utility as molecular adjuvants to enhance vaccine efficacy. The antibodies can be used to increase the protective efficacy of a variety of vaccines, such as those directed against hepatitis B, malaria, HIV.
In the case of vaccination, the anti-ICOS antibody will typically be one that lacks Fc effector function and therefore does not mediate ADCC, CDC or ADCP. The antibody may be provided without an Fc region or with an effector null constant region. Optionally, the anti-ICOS antibody may have a heavy chain constant region that binds to one or more types of Fc receptors but does not induce ADCC, CDC or ADCP activity, or exhibits lower ADCC, CDC and ADCP activity as compared to wild-type human IgG 1. Such constant regions may not be able to bind or may bind with lower affinity to the specific Fc receptor responsible for triggering ADCC, CDC or ADCP activity. Alternatively, the anti-ICOS antibody may comprise a heavy chain constant region that is positive for Fc effector function when cellular effector function is acceptable or desirable in the context of vaccination. For example, any of the IgG1, IgG4, and IgG4.pe formats may be used for anti-ICOS antibodies in vaccination protocols, and other examples of suitable isotypes and antibody constant regions are set forth in more detail elsewhere herein.
Formulations and administration
The antibodies may be monoclonal or polyclonal, but are preferably provided as monoclonal antibodies for therapeutic use. It may be provided as part of a mixture of other antibodies, optionally including antibodies with different binding specificities.
The antibodies and encoding nucleic acids according to the invention will generally be provided in isolated form. Thus, antibodies, VH and/or VL domains and nucleic acids purified according to their natural environment or their environment of production may be provided. Isolated antibodies and isolated nucleic acids will be free or substantially free of materials with which they are naturally associated, such as other polypeptides or nucleic acids with which they are found in vivo, or the environment in which they are prepared when prepared by in vitro recombinant DNA techniques. Optionally, the isolated antibody or nucleic acid is (1) free of at least some other proteins that would normally be found therewith, (2) substantially free of other proteins from the same source, e.g., from the same species, (3) expressed by cells from a different species, (4) has been isolated from at least about 50% of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is operably associated with a polypeptide with which it is not associated in nature (by covalent or non-covalent interactions), or (6) does not occur in nature.
The antibody or nucleic acid may be formulated with a diluent or adjuvant and still be isolated for practical purposes-for example it may be mixed with a carrier if used to coat microtiter plates for use in immunoassays and may be mixed with a pharmaceutically acceptable carrier or diluent when used in therapy. As described elsewhere herein, other active ingredients may also be included in the therapeutic formulation. The antibody may be glycosylated naturally in vivo or by systemic glycosylation of heterologous eukaryotic cells, such as CHO cells, or it may be unglycosylated (e.g., where produced by expression in prokaryotic cells). The present invention encompasses antibodies with modified glycosylation patterns. In some applications, modifications that remove undesirable glycosylation sites or removal of fucose moieties, for example, that increase ADCC function, may be useful [38 ]. In other applications, modifications of galactosylation may be performed in order to modify CDC.
Typically, the isolated product comprises at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. The antibody may be substantially free of proteins or polypeptides or other contaminants found in its natural or production environment that would interfere with its therapeutic, diagnostic, prophylactic, research or other use.
The antibody may have been identified, isolated and/or recovered (e.g., naturally or recombinantly) from a component of its production environment. The isolated antibody may be freed from association with all other components from its production environment, e.g., such that the antibody has been separated from FDA approved or approved standards. Contaminant components of the environment in which they are produced (e.g., produced by recombinant transfected cells) are materials that will typically interfere with research, diagnostic, or therapeutic uses for antibodies, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody will be purified: (1) to greater than 95% by weight of the antibody, as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to the extent sufficient to obtain at least 15N-terminal residues or internal amino acid sequences by using a spinning cup sequencer, or (3) purified to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. Isolated antibodies include antibodies in situ within recombinant cells, as at least one component of the antibody's natural environment will not be present. Typically, however, an isolated antibody or nucleic acid encoding it will be prepared by at least one purification step.
The invention provides therapeutic compositions comprising the antibodies described herein. Therapeutic compositions comprising nucleic acids encoding such antibodies are also provided. Encoding nucleic acids are described in more detail elsewhere herein and include DNA and RNA, e.g., mRNA. In the methods of treatment described herein, the use of nucleic acids encoding antibodies and/or cells containing such nucleic acids may be used as an alternative (or addition) to compositions comprising the antibodies themselves. Cells containing nucleic acid encoding the antibody (optionally, where the nucleic acid is stably integrated into the genome) thus correspond to an agent for therapeutic use in a patient. Nucleic acids encoding anti-ICOS antibodies can be introduced into human B lymphocytes, optionally B lymphocytes from a subject patient and modified in vitro. Optionally, memory B cells are used. Administration of cells containing the encoding nucleic acid to a patient provides a depot of cells capable of expressing anti-ICOS antibodies, which may provide therapeutic benefit over a longer period of time than administration of the isolated nucleic acid or isolated antibody.
The compositions may contain suitable carriers, excipients, and other agents incorporated into the formulations to provide improved transfer, delivery, tolerability, and the like. A plurality ofSuitable formulations can be found in all formulary known to medical chemists: remington's Pharmaceutical Sciences, Mack publishing company (Mack publishing company), Easton, Pa. Such formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, vesicle-containing lipids (cationic or anionic) (e.g., LIPOFECTIN)TM) DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion benzyl wax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing benzyl wax. See also Powell et al, "brief of excipients for parenteral formulations" PDA (1998) < 52:238 th & gt, J Pharm Sci Technol & J Pharm Sci Technol & gt, 311. The composition may comprise an antibody or nucleic acid and a medical injection buffer and/or adjuvant.
The antibody or nucleic acid encoding it may be formulated for the desired route of administration to the patient, e.g., in the form of an injectable liquid (optionally an aqueous solution). Various delivery systems are known and may be used to administer the pharmaceutical compositions of the present invention. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Formulating an antibody for subcutaneous administration typically requires concentrating it to a smaller volume than an intravenous formulation. The high potency of the antibodies according to the invention allows them to be used at sufficiently low doses for subcutaneous formulation practice, with advantages over less potent anti-ICOS antibodies.
The compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered with other bioactive agents. Can be administered systemically or locally.
Pharmaceutical compositions may also be delivered in vesicles, particularly in the form of Liposomes (see Langer (1990) Science 249: 1527-.
In certain instances, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment, air pumps may be used (see Langer, supra; Sefton (1987) CRC review for biomedical engineering reviews (Crit. Ref. biomed. Eng.) 14: 201). In another embodiment, polymeric materials may be used; see Medical Applications of Controlled Release (Controlled Release), Langer and Wise (eds.), CRC Press, Po Calardon, Florida (1974). In yet another embodiment, the controlled release system may be placed close to the target of the composition, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, medical applications for controlled release, supra, vol.2, pages 115-138, 1984).
Injectable formulations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, drip infusions, and the like. These injectable formulations can be prepared by well-known methods. For example, injectable formulations can be prepared, for example, by dissolving, suspending or emulsifying the above-described antibody or a salt thereof in a sterile aqueous or oily medium conventionally used for injection. As the aqueous medium for injection, there are, for example, physiological saline, an isotonic solution containing glucose, other auxiliary agents and the like, which may be mixed with an appropriate solubilizing agent such as alcohol (e.g., ethanol), polyhydric alcohol (e.g., propylene glycol, polyethylene glycol) and the like, a nonionic surfactant [ e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]And the like in combination. As the oily medium, for example, sesame oil, soybean oil, etc. are used, which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared may be filled in an appropriate ampoule. The pharmaceutical compositions of the present invention may be delivered subcutaneously or intravenously using standard needles and syringes. It is envisaged that the treatment will not be limited to clinical use. Thus, subcutaneous injection using a needle-free device is also advantageous. Pen delivery as opposed to subcutaneous deliveryThe delivery device is readily applied to deliver the pharmaceutical composition of the present invention. Such pen delivery devices may be reusable or disposable. Reusable pen delivery devices typically utilize replaceable cartridges containing pharmaceutical compositions. Once all the pharmaceutical composition within the cartridge has been administered and the cartridge emptied, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device may then be reused. In disposable pen delivery devices, there is no replaceable cartridge. In practice, disposable pen delivery devices are pre-filled with a pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded. A variety of reusable pen delivery devices and autoinjector delivery devices are used to subcutaneously deliver the pharmaceutical compositions of the present invention. Examples include, but certainly are not limited to, AUTOPENTM(Owen Mumford, Inc. of Woodstock, UK), DISETRONICTMPen (Disetronic Systems from Bergdorf, Switzerland), HUMALOG MIX 75/25TMPen, HUMALOGTMPen, HUMALIN 70/30TMPen (Eli Lilly and Co., Indianapolis, Ind.) pen, NOVOPENTMI. II and III (NovoNordisk of Copenhagen, Denmark), NOVOPEN JUNIORTM(Novo Nordisk of Copenhagen, Denmark), BDTMPen (Becton Dickinson of Franklin Lakes, N.J.), OPTIPENTM、OPTIPEN PROTM、OPTIPENSTARLETTMAnd OPTICLIKTM(sanofi-aventis, Frankfurt, Germany), to name but a few. Examples of disposable pen delivery devices for subcutaneous delivery of the pharmaceutical compositions of the present invention include, but certainly are not limited to, solotarTMPen (Sanofi-Aventis), FLEXPENTM(Novo Nordisk) and KWIKPENTM(Eli Lilly)。
Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in dosage forms suitable for the unit dose that satisfies the dose of the active ingredient. Such unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, and the like. The aforementioned antibodies are typically included in an amount of about 5mg to about 500mg per unit dose of dosage form; especially in the form of injection, about 5mg to about 100mg and about 10mg to about 250mg of the aforementioned antibody can be contained against other dosage forms.
The antibody, nucleic acid, or composition comprising the same may be contained in a medical container, such as a vial, a syringe, an IV container, or an injection device. In one example, the antibody, nucleic acid, or composition is in vitro, and can be in a sterile container. In one example, a kit comprising an antibody, packaging, and instructions for use in a method of treatment as described herein is provided.
One aspect of the invention is a composition comprising an antibody or nucleic acid of the invention and one or more pharmaceutically acceptable excipients, examples of which are set forth above. By "pharmaceutically acceptable" is meant approved or approvable by a U.S. federal regulatory agency or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans. A pharmaceutically acceptable carrier, excipient, or adjuvant can be administered to a patient with an agent, such as any of the antibodies or antibody chains described herein, and does not destroy the pharmacological activity and is non-toxic when administered at a dose sufficient to deliver a therapeutic amount of the agent.
In some embodiments, the anti-ICOS antibody will be the only active ingredient in the composition according to the invention. Thus, a composition may consist of an antibody or it may consist of an antibody with one or more pharmaceutically acceptable excipients. However, the composition according to the invention optionally comprises one or more additional active ingredients. Detailed descriptions of agents that can be combined with anti-ICOS antibodies are provided elsewhere herein. Optionally, the composition contains multiple antibodies (or encoding nucleic acids) in a combined preparation, e.g., a single formulation comprising an anti-ICOS antibody and one or more additional antibodies. Other therapeutic agents that may need to be administered with an antibody or nucleic acid according to the invention include analgesics. Any such agent or combination of agents may be administered in combination with an antibody or nucleic acid according to the invention, or provided in a composition with the antibody or nucleic acid, whether as a combined or separate formulation. The antibodies or nucleic acids according to the invention can be administered separately and sequentially, or concurrently and optionally as a combined preparation with another therapeutic agent or agents such as those mentioned.
anti-ICOS antibodies for specific therapeutic indications may be combined with recognized standard therapies. Thus, for anti-cancer therapy, antibody therapy may be used in treatment regimens that also include, for example, chemotherapy, surgery, and/or radiation therapy. Radiotherapy can be delivered directly to the diseased tissue or systemically in a single dose or in divided doses.
The multiple compositions may be administered separately or simultaneously. By separately administering is meant that the two compositions are administered at different times, e.g., at least 10 minutes, 20 minutes, 30 minutes, or 10 minutes to 60 minutes apart, or 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours apart. We can also administer the compositions 24 hours apart or even longer apart. Alternatively, two or more compositions may be administered simultaneously, e.g., less than 10 minutes or less than 5 minutes apart. In some aspects, the compositions for simultaneous administration may be administered as a mixture, with or without similar or different timed release mechanisms for each component.
Antibodies and nucleic acids encoding the same may be used as therapeutic agents. The patient herein is generally a mammal, typically a human. The antibody or nucleic acid may be administered to the mammal, for example, by any of the administration routes mentioned herein.
Typically, it is administered in a "therapeutically effective amount," which is an amount that produces the desired effect for which an amount sufficient to exhibit a benefit is administered to the patient. The exact amount will depend on The purpose of The treatment, and will be determinable by one of skill in The Art using known techniques (see, e.g., Lloyd (1999) The Art, Science, and technology of drug Compounding (The Art, Science and technology of Pharmaceutical Compounding)). Prescription of treatment (e.g., determination of dosage, etc.) is within the responsibility of general practitioners and other physicians, and may depend on the severity of the symptoms and/or progression of the disease being treated. A therapeutically effective amount or suitable dose of an antibody or nucleic acid can be determined by comparing its in vitro activity in animal models with its in vivo activity. Methods for extrapolating effective doses to humans in mice and other test animals are known.
As indicated by in vivo studies described in the examples herein, anti-ICOS antibodies may be effective at a range of doses. Pharmacodynamic studies are reported in example 24.
The anti-ICOS antibody may be administered in an amount within one of the following dosage ranges:
from about 10 mug/kg body weight to about 100mg/kg body weight,
about 50 mug/kg body weight to about 5mg/kg body weight,
from about 100 mug/kg body weight to about 10mg/kg body weight,
from about 100 mug/kg body weight to about 20mg/kg body weight,
about 0.5mg/kg body weight to about 20mg/kg body weight, or
About 5mg/kg body weight or less, e.g., less than 4mg/kg, less than 3mg/kg, less than 2mg/kg, or less than 1 mg/kg.
The optimal therapeutic dose for humans may be between 0.1mg/kg and 0.5mg/kg, for example about 0.1mg/kg, 0.15mg/kg, 0.2 mg/kg, 0.25mg/kg, 0.3mg/kg, 0.35mg/kg, 0.4mg/kg, 0.45mg/kg or 0.5 mg/kg. For a fixed dosing regimen for an adult human, a suitable dose may be between 8mg and 50mg or between 8mg and 25mg, for example 15mg or 20 mg.
In the methods of treatment described herein, one or more doses may be administered. In some cases, a single dose may be effective to obtain long-term benefits. Thus, the method may comprise administering a single dose of the antibody, nucleic acid encoding or composition thereof. Alternatively, multiple doses may be administered, typically sequentially and spaced apart by a period of days, weeks or months. The anti-ICOS antibody may be administered to the patient repeatedly at intervals of 4 to 6 weeks, for example, every 4 weeks, every 5 weeks, or every 6 weeks. Optionally, the anti-ICOS antibody may be administered to the patient once a month or less frequently, e.g., every two months or every three months. Thus, a method of treating a patient may comprise administering a single dose of an anti-ICOS antibody to the patient, and no repeat dosing for at least one month, at least two months, at least three months, and optionally at least 12 months.
As discussed in example 11c, similar therapeutic effects can be obtained using one or more doses of anti-ICOS antibody, which can be the result of a single dose of antibody that can effectively reset the tumor microenvironment. The physician can adjust the dosage regimen of the anti-ICOS antibody for the disease and the patient undergoing therapy, taking into account the disease state and any other therapeutic agents or treatments (e.g., surgery, radiation therapy, etc.) that are being combined with the anti-ICOS antibody. In some embodiments, an effective dose of the anti-ICOS antibody is administered more frequently than once a month, e.g., once every three weeks, once every two weeks, or once a week. Treatment with the anti-ICOS antibody may include multiple doses administered over a period of at least one month, at least six months, or at least one year.
As used herein, the term "treatment" or "ameliorating" refers to a therapeutic treatment in which the goal is to reverse, alleviate, ameliorate, inhibit, slow or stop the progression or severity of a condition associated with a disease or disorder. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease, or disorder. Treatment is typically "effective" in the event that one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" in the event that progression of the disease is reduced or halted. That is, "treating" includes not only improving the symptoms or markers, but also terminating or at least slowing the progression or worsening of the symptoms, as compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable. The term "treating" a disease also includes providing relief from the symptoms or side effects of the disease (including palliative treatment). For treatments to be effective, a complete cure is not expected. In certain aspects, the method may also include a cure. In the context of the present invention, the treatment may be a prophylactic treatment.
T cell therapy
WO2011/097477 describes the use of an anti-ICOS antibody for producing and expanding T cells by contacting a population of T cells with a first agent that provides a primary activation signal (e.g., an anti-CD 3 antibody) and a second agent that activates ICOS (e.g., an anti-ICOS antibody), optionally in the presence of a polarizing agent such as IL-1 β, IL-6, neutralizing Th17 against ifny and/or anti-IL-4.
Morphological analysis of anti-ICOS antibodies as therapeutic candidates
It was observed that candidate therapeutic anti-ICOS antibodies were able to induce morphological changes in cells when coupled to a solid surface and contacted with ICOS expressing T cells. Upon addition of ICOS + T cells to wells coated internally with anti-ICOS antibody, the cells were seen to change from their original circular shape, adopt a spindle shape, diffuse and adhere to the antibody-coated surface. This morphological change was not observed with the control antibody. Furthermore, the effect was found to be dose-dependent, with faster and/or more pronounced shape changes occurring with increasing concentration of antibody on the surface. Shape changes provide an alternative indicator that T cells bind to ICOS and/or are agonized by anti-ICOS antibodies. The assay can be used to identify antibodies that promote the multimerization of ICOS on the surface of T cells. Such antibodies represent therapeutic candidate agonist antibodies. Conveniently, the visual indicator provided by such an assay is a simple method of screening for antibodies or cells, particularly large numbers of antibodies or cells. The analysis can be automated to run in a high throughput system.
Accordingly, one aspect of the invention is an assay for selecting antibodies that bind ICOS, optionally for selecting ICOS agonist antibodies, said assay comprising:
providing an array of antibodies immobilized (attached or adhered) to the substrate in the test wells;
adding ICOS-expressing cells (e.g., activated primary T cells or MJ cells) to the test wells;
observing the morphology of the cells;
detecting the change of the shape of the cells from a circle to flattening the substrate tightly attached to the holes; wherein the shape change indicates that the antibody is an antibody that binds ICOS, optionally an ICOS agonist antibody, and
antibodies were selected from the test wells.
Assays may be run in a plurality of test wells, each well containing a different antibody for testing, optionally run in parallel, for example in a 96-well plate format. The substrate is preferably the inner surface of the hole. Thus, a two-dimensional surface is provided against which flattened cells can be observed. For example, the well bottom and/or the well walls may be coated with the antibody. The antibody may be tethered to the substrate by the constant region of the antibody.
Negative controls may be included, such antibodies are known not to bind ICOS, preferably using antibodies that do not bind to antigens on the surface of ICOS-expressing cells. The analysis may comprise quantifying the extent of the morphological change and, where multiple antibodies are tested, selecting an antibody that elicits a greater morphological change than one or more other test antibodies.
The selection antibody may comprise expression of a nucleic acid encoding an antibody present in a test well of interest, or expression of an antibody comprising a CDR or antigen binding domain of an antibody. The antibodies can optionally be reformatted, for example, to provide antibodies comprising the antigen binding domain of the selected antibody, e.g., antibody fragments or antibodies comprising different constant regions. The selected antibody preferably has a human IgG1 constant region or other constant region as described herein. The selected antibodies can be further formulated in compositions comprising one or more additional ingredients-suitable pharmaceutical formulations are discussed elsewhere herein.
Clause and subclause
Examples of the invention are set forth in the following numbered clauses, which are part of the embodiments.
Clause 1. an isolated antibody that binds to the extracellular domain of human and/or mouse ICOS, wherein the antibody comprises: a VH domain comprising an amino acid sequence having at least 95% sequence identity to STIM003 VH domain SEQ ID NO: 408; and a VL domain comprising an amino acid sequence having at least 95% sequence identity to STIM003 VL domain SEQ ID No. 415.
Clause 2. the antibody according to clause 1, wherein the VH domain comprises the heavy chain complementarity determining region (HCDR) repertoire HCDR1, HCDR2 and HCDR3, wherein
HCDR1 is STIM003HCDR1 having the amino acid sequence SEQ ID NO:405,
HCDR2 is STIM003HCDR2 having the amino acid sequence SEQ ID NO:406,
HCDR3 is STIM003HCDR3 having the amino acid sequence SEQ ID NO: 407.
Clause 3. the antibody according to clause 1 or clause 2, wherein the VL domain comprises a light chain complementarity determining region (LCDR) set LCDR1, LCDR2 and LCDR3, wherein
LCDR1 is STIM003LCDR1 having the amino acid sequence SEQ ID NO:412,
LCDR2 is STIM003LCDR2 having the amino acid sequence SEQ ID NO:413,
LCDR3 is STIM003LCDR3 having the amino acid sequence SEQ ID NO: 414.
Clause 4. the antibody according to clause 1, wherein the VH domain amino acid sequence is SEQ ID NO:408, and/or wherein the VL domain amino acid sequence is SEQ ID NO: 415.
Clause 5. an isolated antibody that binds to the extracellular domain of human and/or mouse ICOS comprising an antibody VH domain comprising Complementarity Determining Regions (CDRs) HCDR1, HCDR2, and HCDR3, and
an antibody VL domain comprising complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein
HCDR1 is the HCDR1 of STIM001, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or comprises said HCDR1 with 1, 2, 3, 4 or 5 amino acid alterations,
the HCDR2 is the HCDR2 of STIM001, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008, or STIM009, or comprises the HCDR2 with 1, 2, 3, 4, or 5 amino acid alterations, and/or
HCDR3 is HCDR3 of STIM001, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or comprises said HCDR3 with 1, 2, 3, 4 or 5 amino acid alterations.
Clause 6. the antibody according to clause 5, wherein the antibody heavy chain CDRs are those of STIM001, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or comprise STIM001, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 heavy chain CDRs with 1, 2, 3, 4 or 5 amino acid alterations.
Clause 7. the antibody according to clause 6, wherein the antibody VH domain has the heavy chain CDRs of STIM 003.
Clause 8. an isolated antibody that binds to the extracellular domain of human and/or mouse ICOS comprising an antibody VH domain comprising the complementarity determining regions HCDR1, HCDR2 and HCDR3, and
an antibody VL domain comprising complementarity determining regions LCDR1, LCDR2, and LCDR3,
wherein LCDR1 is LCDR1 of STIM001, STIM002-B, STIM, STIM004, STIM005, STIM006, STIM007, STIM008, or STIM009, or said LCDR1 comprising an alteration of 1, 2, 3, 4, or 5 amino acids,
LCDR2 is the LCDR2 of STIM001, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008, or STIM009, or comprises the LCDR2 with 1, 2, 3, 4, or 5 amino acid alterations, and/or
LCDR3 is LCDR3 of STIM001, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or comprises said LCDR3 with 1, 2, 3, 4 or 5 amino acid alterations.
Clause 9. the antibody according to any one of clauses 5 to 8, wherein the antibody light chain CDRs are those of STIM001, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or comprise STIM001, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 light chain CDRs with 1, 2, 3, 4 or 5 amino acid alterations.
Clause 10. the antibody according to clause 9, wherein the antibody VL domain has the light chain CDRs of STIM 003.
Clause 11. the antibody according to any one of clauses 5 to 10, comprising VH and/or VL domain framework regions of a human germline gene segment sequence.
Clause 12. the antibody according to any one of clauses 5 to 11, comprising a VH domain, which VH domain
(i) Is derived from the recombination of a human heavy chain V gene segment, a human heavy chain D gene segment and a human heavy chain J gene segment, wherein
The V fragment is IGHV1-18 (e.g., V1-18 x 01), IGVH3-20 (e.g., V3-20 x d01), IGVH3-11 (e.g., V3-11 x 01), or IGVH2-5 (e.g., V2-5 x 10);
the D gene segment is IGHD6-19 (e.g., IGHD6-19 x 01), IGHD3-10 (e.g., IGHD3-10 x 01), or IGHD3-9 (e.g., IGHD3-9 x 01); and/or
The J gene segment is IGHJ6 (e.g., IGHJ6 x 02), IGHJ4 (e.g., IGHJ4 x 02), or IGHJ3 (e.g., IGHJ3 x 02), or
(ii) Comprising framework regions FR1, FR2, FR3 and FR4, wherein
FR1 is aligned with human germline V gene segments IGHV1-18 (e.g., V1-18 x 01), IGVH3-20 (e.g., V3-20 x d01), IGVH3-11 (e.g., V3-11 x 01), or IGVH2-5 (e.g., V2-5 x 10), optionally with 1, 2, 3, 4, or 5 amino acid alterations,
FR2 is aligned with human germline V gene segments IGHV1-18 (e.g., V1-18 x 01), IGVH3-20 (e.g., V3-20 x d01), IGVH3-11 (e.g., V3-11 x 01), or IGVH2-5 (e.g., V2-5 x 10), optionally with 1, 2, 3, 4, or 5 amino acid alterations,
FR3 is aligned with human germline V gene segments IGHV1-18 (e.g., V1-18 x 01), IGVH3-20 (e.g., V3-20 x d01), IGVH3-11 (e.g., V3-11 x 01), or IGVH2-5 (e.g., V2-5 x 10), optionally with 1, 2, 3, 4, or 5 amino acid alterations, and/or
FR4 is aligned with human germline J gene segment IGJH6 (e.g., JH6 x 02), IGJH4 (e.g., JH4 x 02), or IGJH3 (e.g., JH3 x 02), optionally with 1, 2, 3, 4, or 5 amino acid alterations.
Clause 13. the antibody according to any one of clauses 5 to 12, comprising an antibody VL domain, which VL domain
(i) Derived from the recombination of a human light chain V gene segment and a human light chain J gene segment, wherein
The V segment is IGKV2-28 (e.g., IGKV2-28 x 01), IGKV3-20 (e.g., IGKV3-20 x 01), IGKV1D-39 (e.g., IGKV1D-39 x 01), or IGKV3-11 (e.g., IGKV3-11 x 01), and/or
The J gene segment is IGKJ4 (e.g., IGKJ4 x 01), IGKJ2 (e.g., IGKJ2 x 04), IGLJ3 (e.g., IGKJ3 x 01), or IGKJ1 (e.g., IGKJ1 x 01); or
(ii) Comprising framework regions FR1, FR2, FR3 and FR4, wherein
FR1 is aligned with human germline V gene segments IGKV2-28 (e.g., IGKV2-28 x 01), IGKV3-20 (e.g., IGKV3-20 x 01), IGKV1D-39 (e.g., IGKV1D-39 x 01), or IGKV3-11 (e.g., IGKV3-11 x 01), optionally with 1, 2, 3, 4, or 5 amino acid alterations,
FR2 is aligned with human germline V gene segments IGKV2-28 (e.g., IGKV2-28 x 01), IGKV3-20 (e.g., IGKV3-20 x 01), IGKV1D-39 (e.g., IGKV1D-39 x 01), or IGKV3-11 (e.g., IGKV3-11 x 01), optionally with 1, 2, 3, 4, or 5 amino acid changes,
FR3 is aligned with human germline V gene segments IGKV2-28 (e.g., IGKV2-28 a 01), IGKV3-20 (e.g., IGKV3-20 a 01), IGKV1D-39 (e.g., IGKV1D-39 a 01), or IGKV3-11 (e.g., IGKV3-11 a 01), optionally with 1, 2, 3, 4, or 5 amino acid alterations, and/or
FR4 is aligned with human germline J gene segments IGKJ4 (e.g., IGKJ4 x 01), IGKJ2 (e.g., IGKJ2 x 04), IGKJ3 (e.g., IGKJ3 x 01), or IGKJ1 (e.g., IGKJ1 x 01), optionally with 1, 2, 3, 4, or 5 amino acid alterations.
Clause 14. an antibody according to any one of clauses 5 to 13, which comprises an antibody VH domain which is the VH domain of STIM001, STIM002-B, STIM003, STIM004 or STIM005, STIM006, STIM007, STIM008 or STIM009, or which has an amino acid sequence at least 90% identical to the antibody VH domain sequence of STIM001, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM 009.
Clause 15, the antibody according to any one of clauses 5 to 14, which comprises an antibody VL domain that is the VL domain of STIM001, STIM002-B, STIM003, STIM004 or STIM005, STIM006, STIM007, STIM008 or STIM009, or which has an amino acid sequence that is at least 90% identical to the antibody VL domain sequence of STIM001, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM 009.
Clause 16. the antibody according to clause 15, comprising
An antibody VH domain selected from the VH domains of STIM001, STIM002-B, STIM003, STIM004 or STIM005, STIM006, STIM007, STIM008 or STIM009, or having an amino acid sequence at least 90% identical to the sequence of the antibody VH domain of STIM001, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, and
an antibody VL domain that is the VL domain of the selected antibody, or that has an amino acid sequence that is at least 90% identical to the antibody VL domain sequence of the selected antibody.
Clause 17. the antibody according to clause 16, comprising a STIM003 VH domain and a STIM003 VL domain.
Clause 18. the antibody according to any one of the preceding clauses, comprising an antibody constant region.
Clause 19. the antibody according to clause 18, wherein the constant region comprises a human heavy chain constant region and/or a light chain constant region.
Clause 20 the antibody according to clause 18 or clause 19, wherein the constant region is Fc effector positive.
Clause 21. the antibody according to clause 20, comprising an Fc region having enhanced ADCC, ADCP and/or CDC function as compared to a native human Fc region.
Clause 22 the antibody according to any one of clauses 18 to 21, wherein the antibody is IgG 1.
Clause 23 the antibody of clause 21 or clause 22, wherein the antibody is afucosylated.
Clause 24. the antibody according to any one of the preceding clauses, conjugated to a cytotoxic drug or prodrug.
Clause 25. the antibody according to any one of the preceding clauses, which is a multispecific antibody.
Clause 26. an isolated antibody that binds the extracellular domain of human and mouse ICOS with an affinity (KD) of less than 50nM as determined by surface plasmon resonance.
Clause 27. the antibody according to clause 26, wherein the antibody binds the extracellular domain of human and mouse ICOS with an affinity (KD) of less than 5nM as determined by surface plasmon resonance.
Clause 28. the antibody according to clause 26 or clause 27, wherein the KD for binding to the extracellular domain of human ICOS is within 10-fold of the KD for binding to the extracellular domain of mouse ICOS.
Clause 29. a composition comprising an isolated antibody according to any one of the preceding clauses and a pharmaceutically acceptable excipient.
Clause 30. a composition comprising an isolated nucleic acid encoding the antibody according to any one of clauses 1 to 28 and a pharmaceutically acceptable excipient.
Clause 31. a method of modulating the balance of regulatory T cells (tregs) and effector T cells (teffs) in a patient to enhance a Teff response, comprising administering to the patient an antibody according to any one of clauses 1 to 28 or a composition according to clause 29.
Clause 32. a method of treating a disease or condition susceptible to therapy by depleting regulatory T cells (tregs) and/or enhancing effector T cell (Teff) responses in a patient, the method comprising administering to the patient an antibody according to any of clauses 1 to 28 or a composition according to clause 29.
Clause 33. the antibody according to any one of clauses 1 to 28 or the composition according to clause 29 for use in a method of treating a human by therapy.
Clause 34. an antibody or composition for use according to clause 33 for use in modulating the balance of regulatory T cells (tregs) and effector T cells (teffs) in a patient to enhance an effector T cell response.
Clause 35. an antibody or composition for use according to clause 33 for use in treating a disease or condition susceptible to therapy by depleting regulatory T cells (tregs) and/or enhancing effector T cell (Teff) responses in a patient.
Clause 36. the method according to clause 32 or the antibody or composition for use according to clause 35, wherein the disease is cancer or a solid tumor.
Clause 37. the antibody according to any one of clauses 1 to 28 or the composition according to clause 29 for use in a method of treating cancer in a human patient.
Clause 38. a method of treating cancer in a human patient comprising administering to the patient an antibody according to any one of clauses 1 to 28 or a composition according to clause 29.
Clause 39. the method or antibody or composition for use according to any one of clauses 36 to 38, wherein the cancer is renal cell carcinoma, head and neck cancer, melanoma, non-small cell lung cancer, or diffuse large B-cell lymphoma.
Clause 40. the method or antibody or composition for use according to any one of clauses 31 to 39, wherein the method comprises administering the antibody and another therapeutic agent and/or radiation therapy to the patient.
Clause 41. the method or antibody or composition for use according to clause 40, wherein the therapeutic agent is an anti-PD-L1 antibody.
Clause 42. the method or antibody or composition for use according to clause 41, wherein the anti-PD-L1 antibody comprises a VH domain having the amino acid sequence SEQ ID NO:299 and a VL domain having the amino acid sequence SEQ ID NO: 300.
Clause 43. the method or antibody or composition for use according to clause 41 or clause 42, wherein the therapeutic agent is an anti-PD-L1 IL-2 immunocytokine.
Clause 44. the method or antibody or composition for use according to clause 43, wherein the anti-PD-L1 antibody is an immunocytokine comprising human wild-type or variant IL-2.
Clause 45. the method or antibody or composition for use according to clause 44, wherein the anti-ICOS antibody and the anti-PDL 1 antibody are each capable of mediating ADCC, ADCP and/or CDC.
Clause 46. the method or antibody or composition for use according to any one of clauses 41 to 45, wherein the anti-ICOS antibody is a human IgG1 antibody and the anti-PDL 1 antibody is a human IgG1 antibody.
Clause 47. the method or antibody or composition for use according to clause 40, wherein the therapeutic agent is an anti-PD-1 antibody.
Clause 48. the method or antibody or composition for use according to clause 40, wherein the other therapeutic agent is IL-2.
Clause 49. the method or antibody or composition for use according to any one of clauses 40 to 48, wherein the method comprises administering the anti-ICOS antibody after administering the additional therapeutic agent and/or radiation therapy.
Clause 50. the method or antibody or composition for use according to any one of clauses 31 to 49, wherein
The anti-ICOS antibody is conjugated to a prodrug, and wherein
The method or use comprises
Administering to the patient an anti-ICOS antibody and
selectively activates the prodrug at the target tissue site.
Clause 51. the method or antibody or composition for use according to clause 50, wherein the patient has a solid tumor and the method comprises selectively activating the prodrug in the tumor.
Clause 52. the method or antibody or composition for use according to clause 50 or clause 51, comprising selectively activating the prodrug by photoactivation.
Clause 53. a combination of an anti-ICOS human IgG1 antibody and an anti-PDL 1 human IgG1 antibody for use in a method of treating cancer in a patient.
Clause 54. a method of treating cancer in a patient, comprising administering to the patient an anti-ICOS human IgG1 antibody and an anti-PD-L1 human IgG1 antibody.
Clause 55. an anti-ICOS antibody for use in a method of treating cancer in a patient, the method comprising administering to the patient an anti-ICOS antibody and an anti-PD-L1 antibody, wherein a single dose of the anti-ICOS antibody is administered.
Clause 56. the anti-ICOS antibody for use according to clause 55, wherein the anti-ICOS antibody is a human IgG1 antibody and the anti-PD-L1 antibody is a human IgG1 antibody.
Clause 57. the combination according to clause 53, the method according to clause 54, or the anti-ICOS antibody for use according to clause 55 or clause 56, wherein the cancer is renal cell carcinoma, head and neck cancer, melanoma, non-small cell lung cancer, or diffuse large B-cell lymphoma.
Clause 58. the method according to any one of clauses 41-46 or 53-54, or the antibody, composition or combination for use, comprising administering to the patient an anti-ICOS antibody and an anti-PD-L1 antibody, wherein a single dose of the anti-ICOS antibody is administered.
Clause 59. the method or antibody, composition or combination for use according to clause 58, wherein the method comprises administering a single dose of the anti-ICOS antibody followed by multiple doses of the anti-PD-L1 antibody.
Clause 60. the method or antibody for use, composition or combination according to any one of clauses 41 to 46 or 53 to 54, wherein the anti-ICOS antibody and the anti-PDL 1 antibody are provided as separate compositions for administration.
Clause 61. the method or antibody for use, composition or combination according to any one of clauses 41-46 or 53-60, wherein the anti-ICOS antibody and/or the anti-PD-L1 antibody comprises a human IgG1 constant region comprising the amino acid sequence SEQ ID NO: 340.
Clause 62. an anti-ICOS antibody for use in a method of treating a patient, the method comprising administering the anti-ICOS antibody to a patient having increased ICOS-positive regulatory T cell content following treatment with another therapeutic agent.
Clause 63. a method of treating a patient, the method comprising administering an anti-ICOS antibody to a patient having increased ICOS-positive regulatory T cell content following treatment with another therapeutic agent.
Clause 64. the anti-ICOS antibody for use according to clause 62 or the method according to clause 63, wherein the method comprises administering to the patient a therapeutic agent, determining that the patient has increased ICOS-positive regulatory T cell content after treatment with the agent, and administering to the patient an anti-ICOS antibody to reduce the regulatory T cell content.
Clause 65. the anti-ICOS antibody for use or the method according to any one of clauses 62 to 64, wherein the therapeutic agent is IL-2 or an immunomodulatory antibody (e.g., anti-PDL-1, anti-PD-1, or anti-CTLA-4).
Clause 66. the anti-ICOS antibody for use according to any one of clauses 62 to 65, wherein the method comprises treating a tumor, e.g., melanoma, such as metastatic melanoma.
Clause 67. an anti-ICOS antibody for use in a method of treating cancer in a patient by in vivo vaccination of cancer cells in the patient, the method comprising
Treating the patient with a therapy that causes immune cell death of the cancer cells such that the antigen is presented to antigen-specific effector T cells, and
administering an anti-ICOS antibody to the patient, wherein the anti-ICOS antibody enhances an antigen-specific effector T cell response.
Clause 68. a method of treating cancer in a patient by in vivo vaccination of cancer cells in the patient, the method comprising
Treating the patient with a therapy that causes immune cell death of the cancer cells such that the antigen is presented to antigen-specific effector T cells, and
administering an anti-ICOS antibody to the patient, wherein the anti-ICOS antibody enhances an antigen-specific effector T cell response.
Clause 69.a method of treating cancer in a patient by in vivo vaccination of cancer cells in the patient, the method comprising administering to the patient an anti-ICOS antibody, wherein
The patient has been previously treated with a therapy that causes immune cell death of cancer cells, such that antigen is presented to antigen-specific effector T cells, and wherein
anti-ICOS antibodies enhance antigen-specific effector T cell responses.
Clause 70. the anti-ICOS antibody for use or the method according to any of clauses 67 to 69, wherein the therapy that causes immune cell death is radiation cancer cells, administration of a chemotherapeutic agent and/or administration of an antibody directed against a tumor-associated antigen.
Clause 71. the anti-ICOS antibody for use or the method according to clause 70, wherein the chemotherapeutic agent is oxaliplatin (oxaliplatin).
Clause 72, the anti-ICOS antibody for use or the method of clause 70, wherein the tumor-associated antigen is HER2 or CD 20.
Clause 73. an anti-ICOS antibody for use in a method of treating cancer in a patient, wherein the cancer is or has been characterized as positive for expression of ICOS ligand and/or FOXP 3.
Clause 74. a method of treating cancer in a patient, wherein the cancer is or has been characterized as positive for expression of ICOS ligand and/or FOXP3, the method comprising administering to the patient an anti-ICOS antibody.
Clause 75. an anti-ICOS antibody for use according to clause 73 or a method according to clause 74, wherein the method comprises:
testing a sample from the patient to determine that the cancer expresses ICOS ligand and/or FOXP 3;
selecting a patient for treatment with an anti-ICOS antibody; and
the anti-ICOS antibody is administered to the patient.
Clause 76. the anti-ICOS antibody for use according to clause 73 or the method according to clause 74, wherein the method comprises administering the anti-ICOS antibody to a patient whose test sample indicates that the cancer is positive for expression of ICOS ligand and/or FOXP 3.
Clause 77 the anti-ICOS antibody or method for use according to clause 75 or clause 76, wherein the sample is a biopsy sample of a solid tumor.
Clause 78. an anti-ICOS antibody for use in a method of treating cancer in a patient, wherein the cancer is or has been characterized as refractory to treatment with an immunooncology drug, e.g., an anti-CTLA-4 antibody, an anti-PD 1 antibody, an anti-PD-L1 antibody, an anti-CD 137 antibody, or an anti-GITR antibody.
Clause 79.a method of treating cancer in a patient, wherein the cancer is or has been characterized as refractory to treatment with an immunooncology agent, e.g., an anti-CTLA-4 antibody, an anti-PD 1 antibody, an anti-PD-L1 antibody, an anti-CD 137 antibody, or an anti-GITR antibody, the method comprising administering to the patient an anti-ICOS antibody.
Clause 80. an anti-ICOS antibody for use according to clause 78 or a method according to clause 79, wherein the method comprises:
treating the patient with an immunooncology drug;
determining that the cancer is not responsive to the drug;
selecting a patient for treatment with an anti-ICOS antibody; and
the anti-ICOS antibody is administered to the patient.
Clause 81. the anti-ICOS antibody for use according to clause 78 or the method according to clause 79, wherein the method comprises administering the anti-ICOS antibody to a patient whose cancer is refractory to prior treatment with the immunooncology drug.
Clause 82, the anti-ICOS antibody for use or the method according to any one of clauses 73 to 81, wherein the cancer is a tumor derived from a cell capable of acquired expression of ICOS ligand.
Clause 83. the anti-ICOS antibody for use or the method according to clause 82, wherein the cancer is melanoma.
Clause 84. the anti-ICOS antibody for use or the method according to any one of clauses 73 to 81, wherein the cancer is derived from an antigen presenting cell, such as a B lymphocyte (e.g., a B cell lymphoma, such as diffuse large B cell lymphoma) or a T lymphocyte.
Clause 85. the anti-ICOS antibody for use or the method according to any one of clauses 73 to 81, wherein the cancer is resistant to treatment with the anti-CD 20 antibody.
Clause 86, the anti-ICOS antibody for use or the method according to clause 85, wherein the cancer is B-cell lymphoma.
Clause 87. the anti-ICOS antibody for use or the method according to clause 86, wherein the anti-CD 20 antibody is rituximab.
Clause 88 the anti-ICOS antibody for use or the method according to any one of clauses 85 to 87, wherein the method comprises treating the patient with an anti-CD 20 antibody;
determining that the cancer is not responsive to the anti-CD 20 antibody;
testing a sample from the patient to determine that the cancer expresses ICOS ligand;
selecting a patient for treatment with an anti-ICOS antibody; and
the anti-ICOS antibody is administered to the patient.
Clause 89 the anti-ICOS antibody for use or the method according to any one of clauses 85 to 87, wherein the method comprises administering the anti-ICOS antibody to a patient whose cancer is refractory to prior treatment with the anti-CD 20 antibody.
Clause 90 the anti-ICOS antibody for use or the method according to any one of clauses 67 to 89, wherein the cancer is a solid tumor.
Clause 91. the anti-ICOS antibody for use or the method according to any one of clauses 67 to 89, wherein the cancer is a hematological liquid tumor.
Clause 92. the anti-ICOS antibody for use or the method according to clause 90 or 91, wherein the regulatory T cells of the tumor are higher.
Clause 93. an anti-ICOS antibody for use or a method according to any of clauses 53 to 92, wherein the anti-ICOS antibody is as defined in any of clauses 1 to 28 or provided in the form of a composition according to clause 29.
Clause 94. a transgenic non-human mammal having a genome comprising a human or humanized immunoglobulin locus encoding a human variable region gene segment, wherein the mammal does not express ICOS.
Clause 95. a method of generating an antibody that binds to the extracellular domain of human and non-human ICOS, comprising
(a) Immunizing a mammal according to clause 94 with a human ICOS antigen;
(b) isolating antibodies produced by the mammal;
(c) testing the ability of the antibody to bind to human ICOS and non-human ICOS; and
(d) one or more of human and non-human ICOS are selected to bind simultaneously.
Clause 96. the method according to clause 95, comprising immunizing the mammal with a cell expressing human ICOS.
Clause 97. the method according to clause 95 or clause 96, comprising
(c) Testing the ability of the antibody to bind human ICOS and non-human ICOS using surface plasmon resonance and determining binding affinity; and
(d) one or more are selected that have a KD for binding to human ICOS of less than 50nM and a KD for binding to non-human ICOS of less than 500 nM.
Clause 98. the method according to clause 97, comprising
(d) One or more are selected that have a KD for binding to human ICOS of less than 10nM and a KD for binding to non-human ICOS of less than 100 nM.
Clause 99. the method according to any one of clauses 95 to 98, comprising
(c) Testing the ability of the antibody to bind human ICOS and non-human ICOS using surface plasmon resonance and determining binding affinity; and
(d) selecting one or more that have a KD for binding to human ICOS that is within 10-fold of the KD for binding to non-human ICOS.
Clause 100. the method according to clause 99, comprising
(d) Selecting one or more that have a KD for binding to human ICOS that is within 5-fold of the KD for binding to non-human ICOS.
Clause 101. the method according to any one of clauses 95 to 100, comprising testing the ability of an antibody to bind to non-human ICOS from the same species, such as a mammal.
Clause 102. the method according to any one of clauses 95 to 101, comprising testing the ability of an antibody to bind to non-human ICOS from a different species, such as a mammal.
Clause 103. the method according to any one of clauses 95 to 102, wherein the mammal is a mouse or a rat.
Clause 104. the method of any one of clauses 95 to 103, wherein the non-human ICOS is mouse ICOS or rat ICOS.
Clause 105. the method according to any one of clauses 95 to 104, wherein the human or humanized immunoglobulin locus comprises a human variable region gene segment upstream of an endogenous constant region.
Clause 106. the method according to clause 105, comprising
(a) Immunizing a mammal according to clause 94 with a human ICOS antigen, wherein the mammal is a mouse;
(b) isolating antibodies produced by the mouse;
(c) testing the ability of the antibody to bind human ICOS and mouse ICOS; and
(d) one or more antibodies that bind both human and mouse ICOS are selected.
Clause 107. the method according to any one of clauses 95 to 106, comprising isolating nucleic acid encoding the antibody heavy chain variable domain and/or the antibody light chain variable domain.
Clause 108. the method according to any one of clauses 95 to 107, wherein the mammal produces the antibody by recombination of human variable region gene segments and endogenous constant regions.
Clause 109. the method according to clause 107 or clause 108, comprising conjugating a nucleic acid encoding a heavy chain variable domain and/or a light chain variable domain to a nucleotide sequence encoding a human heavy chain constant region and/or a human light chain constant region, respectively.
Clause 110. the method according to any one of clauses 107 to 109, comprising introducing the nucleic acid into a host cell.
Clause 111. the method according to clause 110, comprising culturing the host cell under conditions to express the antibody or the antibody heavy chain variable domain and/or the light chain variable domain.
Clause 112. an antibody or antibody heavy chain variable domain and/or light chain variable domain produced by the method according to any one of clauses 95 to 111.
Clause 113. a method of selecting an antibody that binds ICOS, optionally for use in selecting an ICOS agonist antibody, the assay comprising:
providing an array of antibodies immobilized (attached or adhered) to the substrate in the test wells;
adding ICOS-expressing cells (e.g., activated primary T cells or MJ cells) to the test wells;
observing the cell morphology;
detecting the change of the shape of the cells from a circle to flattening the substrate tightly attached to the holes; wherein the shape change indicates that the antibody is an antibody that binds ICOS, optionally an ICOS agonist antibody;
antibodies were selected from the test wells.
Expressing nucleic acids encoding the CDRs of the selected antibody; and
the antibodies are formulated into compositions comprising one or more additional components.
Various other aspects and embodiments of the invention will be apparent to those skilled in the art in view of this disclosure. All documents referred to in this specification, including any patents or patent applications referred to by published U.S. associates, are incorporated herein by reference in their entirety.
Experimental examples
The following examples describe the generation, characterization and potency of anti-ICOS antibodies. Using KymouseTM(transgenic mouse platform capable of producing antibodies with human variable domains) antibodies were produced. From KymouseTMThe antibody of (a) has a human variable domain produced from human v (d) and J fragments and a mouse constant domain. Endogenous mouse variable genes have been silenced and comprise a very small fraction of the repertoire (less than 0.5% of all heavy chain variable regions are of mouse origin). KymouseTMThe system is described in Lee et al 2014[39 ]]WO2011/004192, WO2011/158009 and WO 2013/061098. This project uses KymouseTMHK strain, wherein the heavy chain locus and the light chain kappa locus are humanized.
ICOS knock-out KymouseTMUsing ICOS proteinsImmunizations were performed with a combination of alternating boosts of proteins and cells expressing human and mouse ICOS.
Hits that bound to human ICOS were identified. The primary selection criteria for screening were binding to ICOS expressing human cells (CHO cells) and binding to ICOS protein (HTRF). Binding to mouse ICOS protein and ICOS expressing mouse cells (CHO cells) was also assessed and taken into account when selecting primary screening hits. Secondary screening was performed using these standard hits. In secondary screening hits were confirmed by determining binding to human and mouse ICOS expressed on CHO cells using flow cytometry.
Based on the large number of antibodies screened, the facet regions that bound to human/cynomolgus and mouse ICOS were identified as determined by surface plasmon resonance and flow cytometry. These antibodies include STIM001, STIM002 and variants thereof STIM002-B, STIM003, STIM004, and STIM 005. An additional four antibodies, STIM006, STIM007, STIM008 and STIM009, were also selected, exhibiting less cross-reactivity with mouse ICOS but agonism with the human ICOS receptor. The data presented here indicate the ability of anti-ICOS antibodies to act as agonists of the ICOS receptor in the ICOS positive CD4+ cell line, and also to exhibit cell killing ability in ADCC assays and the ability to promote anti-tumor immune responses in vivo in primary T cell-based assays.
Example 1: generation of ICOS knockout mice
ICOS knock-out KymouseTMBy using a gene in KymouseTMHK ES cells were produced by homologous recombination. Briefly, a 3.5kb targeting vector encoding puromycin selection was targeted into ES cells. Successful targeting resulted in the replacement of a small region (72bp) of the mouse ICOS locus with a puromycin cassette, thereby interfering with the signal peptide/start codon of the gene. Positive ES clones were amplified and microinjected into mouse blastocysts and raised chimeras were produced to ultimately produce homozygous animals with both humanized heavy and kappa immunoglobulin loci and modified loss-of-function ICOS loci.
Example 2: antigen and cell line preparation
Generation of stably transfected MEF and CHO-S cells expressing human or mouse ICOS
The full-length DNA sequences encoding human and mouse ICOS were ordered as synthetic string DNA and cloned into an expression vector under the control of the CMV promoter for optimization for mammalian expression, and flanked by 3 'and 5' piggyBac-specific terminal repeats that contribute to codons stably integrated into the cell genome (see [40 ]). The expression vector contains a puromycin selection cassette to facilitate stable cell line production. To generate human and mouse ICOS expressing cell lines, respectively, human or mouse ICOS expressing plasmids were co-transfected into Mouse Embryonic Fibroblast (MEF) cell lines and CHO-S cells using FreeStyle Max transfection reagent (Invitrogen) according to the manufacturer' S instructions with plasmids encoding piggyBac transposase. MEF cells were generated from embryos obtained from female mice crossed 129S5 with C57BL 6. Twenty-four hours after transfection, the medium was supplemented with puromycin and grown for at least two weeks to select stable cell lines. Cell culture medium was replaced every 3-4 days. Expression of human or mouse ICOS protein was assessed by flow cytometry using anti-human or anti-mouse ICOS-PE conjugated antibodies (eBioscience), respectively. The complete MEF Medium consists of Dulbecco's modified eagle's Medium (Gibbeco) supplemented with 10% v/v fetal bovine serum (Gibco). The complete CHO-S medium consisted of CD-CHO medium (Gidbaceae) supplemented with 8mM Glutamax (Gibbidae). CHO-S cells are CHO-3E7 cell lines including the pTT5 system available from the National Research Council of Canada, although other CHO cell lines may be used.
Preparation of MEF cells for immunization of mice
The cell culture medium was removed and the cells were washed once with 1 × PBS. Cells were trypsinized for 5 minutes to loosen the cells from the tissue culture surface. Cells were harvested and trypsin was neutralized by addition of complete medium containing 10% v/v Fetal Calf Serum (FCS). The cells were then centrifuged at 300g for 10min and washed with 25ml 1 XPBS. Cells were counted and resuspended at the correct concentration in 1 × PBS.
Cloning and expression of recombinant proteins
Synthetic DNA encoding the extracellular domains of human ICOS (NCBIID: NP-036224.1), mouse ICOS (NCBIID: NP-059508.2), and cynomolgus ICOS (GenBank ID: EHH55098.1) were cloned into pREP4 (Invitrogen) or pTT5 (Canada national research Committee) expression plasmids using standard molecular biology techniques. The constructs also contained a human Fc, mouse Fc, or FLAG His peptide motif to aid in purification and detection. These are added to the DNA construct by overlap extension. All constructs were sequenced prior to expression to ensure their correct sequence composition.
Example 3: immunization
ICOS knock-out of HK kyrice according to the protocol shown in Table E3TM(see example 1), KymouseTMWild type HK strain and KymouseTMWild type HL strain immunization. KymouseTMWild-type HK and HL strains express wild-type mouse ICOS. In the HK strain, the immunoglobulin heavy chain locus and the light chain kappa locus are humanized, and in the HL strain, the immunoglobulin heavy chain locus and the light chain lambda locus are humanized.
TABLE E3. for KymouseTMImmunization protocol for strains
Keywords of the table:
mouse ICOS protein with human Fc
human ICOS protein with human Fc
mICOS MEF-mouse ICOS expressed on MEF cells
hICOS MEF ═ human ICOS expressed on MEF cells
mICOS Fc + hICOS Fc is administered simultaneously to mouse ICOS protein with human Fc + human ICOS protein with human Fc
mICOS MEF + hICOS MEF ═ Simultaneous administration of mouse ICOS expressed on MEF cells + human ICOS expressed on MEF cells
ICOS KO ═ ICOS knock-out HK Kymouse
HK and HL ═ wild-type Kymouse HK and HL genotypes
RIMMS is a modified subcutaneous immunization process (rapid immunization at multiple sites); modification after Kilparick et al [41 ]). The-resting-boosting protocol KM103 and KM111 were initiated by intraperitoneal (i.p.) administration. The Sigma Adjuvant System (Sigma Adjuvant System) is used for all immunotherapies and the rest interval is typically between 2 and 3 weeks. Final boosting is given intravenously in the absence of adjuvant.
Sera from serial or terminal blood samples were analyzed by flow cytometry for the presence of specific antibodies and titration data (where possible) were used to select mice to be used for B cell sorting.
Example 4: comparison of serum titrations between ICOS KO and wild type mice
Serum titrations of immune ICOS KO and immune wild-type Kymouse were determined using flow cytometry. In ICOS KO mice, immunization with the human ICOS antigen induced a serum immunoglobulin response, in which Ig bound to both human and mouse ICOS expressed on CHO cells (fig. 1 a). In contrast, in wild-type Kymouse (expressing mouse ICOS), immunization with the same human ICOS antigen produced sera that showed greatly reduced binding to mouse ICOS compared to the binding of the same sera to human ICOS (fig. 1 b).
Method of producing a composite material
CHO-S cells expressing human ICOS or mouse ICOS (see example 2) or untransfected CHO-S cells (called Wild Type (WT)) suspended in FACS buffer (PBS + 1% w/v BSA + 0.1% w/v sodium azide) at 10%5The density of individual cells/well was distributed to 96 well V-bottom plates (Greiner). Mouse serum titrations were prepared and samples were diluted in FACS buffer. This aliquot, 50 microliters/well, was then added to the cell culture plate. To determine the change in the extent of activity due to immunization, serum from each animal was diluted to 1/100 in FACS buffer and added to the cells at 50 μ l/well prior to immunization. The cells were incubated at 4 ℃ for 1 hour. Cells were washed twice with 150 μ L PBS, centrifuged after each washing step and the supernatant aspirated (centrifuged at 300 × g for 3 min). To detect antibody binding, APC goat-anti-mouse IgG (Jackson ImmunoResearch) was diluted 1/500 in FACS buffer and 50 μ L was added to the cells. In some cases, AF647 goat-anti-mouse IgG was used (jackson immunization study). Cells were incubated at 4 ℃ for 1 hour in the dark, then washed twice with 150 μ L PBS as above. To fix the cells, 100 μ L of 2% v/v paraformaldehyde was added and the cells were incubated for 30 minutes at 4 ℃. The cells were then pelleted by centrifugation at 300 Xg and the plates were resuspended in 50. mu.L of FACS buffer. Fluorescence signal intensity (geometric mean) was measured by flow cytometry using a BD FACS Array instrument.
Example 5: sorting antigen-specific B cells by FACS
B cells expressing anti-ICOS antibodies were recovered from immunized mice using a technique substantially as described in example 1 of WO 2015/040401. Briefly, spleen cells and/or lymph node cells isolated from the immunization protocol were stained with an antibody cocktail containing markers for selection of target cells (CD19), while unwanted cells were excluded from the final sorted population (IgM, IgD, 7 AAD). CD19+B cells were further labeled with fluorescently labeled human ICOS ECD-Fc dimer and fluorescently labeled mouse ICOS ECD-Fc to detect anti-ICOS antibody-producing B cells. Human and mouse ICOS were fluorescently labeled with AlexaFluor647 and AlexaFluor488, respectively-see example 6. Cells were selected that bound human ICOS or both human and mouse ICOS. These cells were single cells sorted by FACS into lysis buffer. V region sequences were recovered using RT-PCR and two additional rounds of PCR, then bridged to the mouse IgG1 constant region and expressed in HEK293 cells. Supernatants from HEK293 cells were screened for presenting ICOS binding and functional antibodies. This method is hereinafter referred to as BCT.
Example 6: screening of antibodies according to BCT
HTRF screening of BCT supernatant for binding to recombinant human and mouse ICOS-Fc
Supernatants collected according to BCT in example 5 were screened for the ability of secreted antibodies to bind human ICOS Fc and mouse ICOS Fc expressed as recombinant proteins. By passing(homogeneous time-resolved fluorescence, Cisbio) assay format647H (Innova Biosciences) labelled ICOS (for use with Innova Biosciences)647H-labeled human ICOS and mouse ICOS, referred to herein as 647hICOS or647 mICOS, respectively) to recognize the binding of secreted antibodies to recombinant human and mouse ICOS. mu.L of BCT supernatant was transferred to a white 384 well small volume non-binding surface polystyrene culture plate (Gelina). 5 μ L of 20nM 647hICOS or647 mICOS diluted in HTRF assay buffer was added to all wells. For human ICOS binding assays, the reference antibody was diluted to 120nM in BCT medium (gibidae # a14351-01) and 5 μ Ι _ was added to the plate. For negative control wells used for human ICOS binding assays, 5 μ L of mouse IgG1 (sigma M9269, referred to as CM7 in some cases) was diluted to 120nM in BCT medium. For mouse ICOS bindingAssay for analysis, reference antibody was diluted to 120nM in BCT medium (gibco # a14351-01) and 5 μ Ι _ was added to the plate. Rat IgG2b isotype control (antin) was added to negative control wells (antin) diluted to 120nM in BCT medium and 5 μ Ι _ was added to the plates. Binding of secreted antibodies to human ICOS was detected by the addition of 10 μ L of goat anti-mouse IgG (Southern biotechnology) directly labeled with europium cryptate (Cisbio) diluted with 1/2000 in HTRF assay buffer. For mouse ICOS binding assays, 5. mu.L of mouse anti-rat IgG2b-UBLB (southern Biotechnology) was added to the positive and negative control wells, and 5. mu.L of HTRF assay buffer was added to all other wells of the plate. Binding was then detected by the addition of 5 μ L goat anti-mouse IgG (southern biotechnology) directly labeled with europium cryptate (Cisbio) diluted in HTRF assay buffer 1/1000. The plates were left to incubate in the dark for 2 hours before reading the time-resolved fluorescence at emission wavelengths of 620nm and 665nm using an Envision plate reader (Perkin Elmer).
The data was analyzed by calculating 665/620 ratio and% effectiveness for each sample according to equation 2 and equation 1, respectively.
For KM103 and KM11-B1, primary hits were selected for binding to human and mouse ICOS based on greater than or equal to 5% effect. For KM135, primary hits were selected for binding to human and mouse ICOS based on greater than or equal to 10% effect. For KM111-B2, the primary hits were defined as greater than or equal to 4% effect for binding to human ICOS and greater than or equal to 3% effect for binding to mouse ICOS.
Equation 1: calculated effect according to primary screen Envision cell binding and HTRF%
Using either the pore ratio value (equation 3) or the 665/620nm ratio (see equation 2) (HTRF)
Non-specific binding-value of wells containing isotype control mouse IgG1
Total binding-value of wells containing reference antibody
Equation 2: 665/620 ratio calculation
665/620 ratio (sample 665/620nm value) × 10,000
Equation 3: calculation of 647/FITC ratio
Data for cell number were primary normalized by dividing mAb channel (647) by FITC (cell dye) channel to obtain "pore ratio value":
screening of BCT supernatants for binding to cells expressing human and mouse ICOS
Supernatants collected from BCT in example 5 were screened for the ability of secreted antibodies to bind to human or mouse ICOS expressed on the surface of CHO-S cells. To determine CHO-S human and mouse ICOS binding, cells were plated at 4X 10 in 384-well black-walled, clear-bottomed tissue culture treated plates (Perkin Elmer)4One/well was inoculated in F12 medium (gibidae) supplemented with 10% FBS (gibidae) and cultured overnight. Media was removed from 384 well assay plates. At least 50 μ L of BCT supernatant in BCT medium at 2 μ g/mL or 50 μ L of reference antibody or isotype IgG1 control antibody diluted in BCT medium (in some cases referred to as Cm7, sigma M9269, final concentration of 2 μ g/mL) was added to each well. The plates were incubated at 4 ℃ for 1 hour. The supernatant was aspirated and 50 μ L of goat anti-mouse 647 (Jackson Immunity study) and 1/500 bright green DNA dye (Life Technologies) diluted in secondary antibody buffer (1 XPBS + 1% BSA + 0.1% sodium azide) at 5 μ g/ml were added to detect antibody binding and visualize cells. The plates were incubated at 4 degrees for 1 hour. The supernatant was aspirated and 25 μ L of 4% v/v paraformaldehyde was added and the plates were incubated at room temperature for 15 minutes. The plates were washed twice with 100 μ L PBS and then the wash buffer was completely removed. Fluorescence intensity was measured using an Envision plate reader (perkin elmer) measuring FITC (excitation 494nm, emission 520nm) and alexafluor647 (excitation 650nm, emission 668 nm). The assay signal was determined as described in equation 3 and the% effect was determined as in equation 1. The total binding was defined using a reference antibody at a final assay concentration of 2 μ g/mL. Non-specific binding was defined using a mouse IgG1 isotype control (sigma) at a final assay concentration of 2 μ g/mL. The criteria for hit selection are based on the analysis signal and% effect.
For KM103, KM111-B1, and KM135, primary hits were selected based on greater than or equal to 10% effect. For KM111-B2, the primary hits were selected based on greater than or equal to 4% effect.
Summary of Primary screening results
| Experiment ID | Supernatant from the screening | Selected first hit |
| KM103 | 1232 | 40 |
| KM111-B1 | 1056 | 198 |
| KM111-B2 | 1056 | 136 |
| KM135 | 704 | 31 |
Table E6. summarizes the number of BCT supernatants from the immunoscreens and the number of supernatants that meet the primary screening selection criteria for binding to human and mouse ICOS.
FACS screening for binding to cells expressing human and mouse ICOS
The BCT supernatants and HEK293 expressing antibodies from example 5 were tested for their ability to bind CHO-S cells expressing human or mouse ICOS.
CHO-S cells expressing human or mouse ICOS (see example 2) were diluted in FACS buffer (PBS 1% BSA 0.1% sodium azide) and treated at 1X 105The density of individual cells/well was distributed to a 96-well V-bottom plate (geliana). Cells were washed with 150 μ L PBS and centrifuged at 300g for 3 min. For supernatant screening, the supernatant was aspirated and 150 μ LPBS was added. This wash step was repeated. To the washed cells, 30. mu.L of the reference or control antibody was added without BCT dilution supernatant or 50. mu.L diluted to 5. mu.g/ml in BCT medium. The cells were incubated at 4 ℃ for 60 minutes. Add 150 μ LFACS buffer and wash cells as described above. To detect antibody binding, 50 μ L of goat-anti-mouse APC (Jackson Immunity study) diluted to 2 μ g/ml in FACS buffer was added to the cells. The cells were incubated at 4 ℃ for 60 minutes. Cells were washed twice with 150 μ L FACS buffer, centrifuged at 300g for 3 minutes after each washing step and the supernatant aspirated. Cells were fixed by adding 25 μ Ι _ of 4% paraformaldehyde for 20 minutes at room temperature. Cells were washed once as above and resuspended in FACS buffer for analysis. Detection by flow cytometry using a BD FACS Array instrumentMeasure APC signal intensity (geometric mean). Data were plotted as geometric means without further calculation.
In this further screen, a small subset of antibodies were selected based on meeting more stringent criteria for species cross-reactivity, as compared to the primary screen. Briefly, the method comprises the following steps:
according to KM103, 4antibodies were selected by taking geometric mean of hybridization controls bound to hICOS, mICOS and WT CHO cells and recognizing > 4-fold more mouse and human binders. These 4antibodies were designated STIM001, STIM002-B, STIM007 and STIM 009.
For KM111-B1, 4antibodies were selected by taking geometric mean values of negative controls (Amanian hamster: clone HTK888) that bind to hICOS, mICOS, and WT CHO cells and recognizing > 10-fold more mouse and human binders.
For KM111-B2, 4antibodies were selected by taking geometric mean values of negative controls (Amanian hamster: clone HTK888) that bind to hICOS, mICOS, and WT CHO cells and recognizing > 4-fold more mouse and human binders. These 4antibodies include STIM003, STIM004 and STIM 005.
For KM135, non-cross-reactive antibodies were identified. Due to technical failures of FACS secondary screening methods, SPR and HTRF were also used for screening, but no antibodies were found that met the desired level of cross-reactivity.
In summary, according to the different multiple immunization protocols described in example 3, ascending 4000BCT supernatants (from ICOS KO mice only) were screened for binding to human ICOS and mouse ICOS, and candidate groups including STIM001, STIM002-B, STIM003, STIM004, STIM005, STIM007, and STIM009 were identified as having the most promising features for further development. These antibodies were used for more detailed characterization.
Separately, two antibodies, STIM006 and STIM008, which did not meet the species cross-reactivity criteria, were also selected for further characterization based on their ability to bind human ICOS.
Example 7: affinity determination by Surface Plasmon Resonance (SPR)
ProteOn XPR36 (BioRad) was used to generate Fab affinity for ICOS leads by SPR. Three anti-human IgG antibodies (Jackson laboratories 109-. Human Fc-labeled human ICOS (hICOS) and mouse ICOS (mICOS) were captured separately on anti-human IgG surface, and purified Fab was used as analyte at 5000nM, 1000nM, 200nM, 40nM and 8nM except for STIM003 used at 1000nM, 200nM, 40nM, 8nM and 2 nM. Buffer injection (i.e., 0nM) was used for dual reference binding sensorgrams and the data was fitted to the 1:1 model inherent to the ProteOn XPR36 analytical software. The analysis was performed at 25 ℃ and using HBS-EP as the handling buffer.
Table E7-1. affinity and kinetic data for selected antibodies as measured by SPR.
In addition, the antibody-antigen binding affinities were compared at different pH values. As previously described, dimeric human ICOS protein, presented as the extracellular domain of ICOS fused to a human Fc region, was captured on an anti-human Fc capture surface formed using a mixture of 3 antibodies, immobilized on a GLC biosensor chip by primary amine coupling. SPR analysis of recombinantly expressed anti-ICOSFab was performed on the ProteOn XPR36 Array system (Burley). Fab fragments were used as analytes to generate binding sensorgrams, which were double referenced using buffer injection (i.e., 0 nM). Subsequent referenced sensorgrams were fitted to the 1:1 model inherent to the ProteOn analysis software. Table E7-2 shows the affinity and kinetic data for the antibodies, all run at 37 ℃ as indicated using HBS-EP at pH 7.4/7.6 or pH 5.5 unless specified otherwise. The data were fitted to a 1:1 model. It should be noted that the data for STIM002 (the affinity of this antibody) that fit poorly to the 1:1 model at both pH 7.4 and 5.5 may therefore be lower than indicated in the table.
Table E7-2 the relative affinities of STIM001, STIM002-B and STIM003Fab for recombinant human ICOS at 37 ℃ except where specified.
Comparison of affinity data at different pH values indicates that the antibody remains bound to its target over a physiological pH range. The tumor microenvironment can be relatively acidic compared to blood, thus maintaining affinity at lower pH is a potential advantage of increasing depletion of T-reg in tumors in vivo.
Example 8: ICOS ligand binding to ICOS receptor neutralization by HTRF analysis
Selected anti-ICOS antibodies were further assessed for their ability to bind ICOS with ICOS ligand (B7-H2) using Homogeneous Time Resolved Fluorescence (HTRF). Mabs of human IgG 1and human IgG4.pe (null effector) isotypes were assessed in:
-HTRF analysis for neutralizing human B7-H2 binding to human ICOS; and
HTRF analysis for neutralizing mouse B7-H2 binding to mouse ICOS.
anti-ICOS antibody C398.4A (hamster IgG in each case) was included for comparison.
A variety of antibodies were found to have high neutralizing potency against human and/or mouse ICOS receptor-ligand binding, and the results indicated that some of these antibodies showed good cross-reactivity. Antibody isotypes had no significant effect, and the difference in results between IgG 1and IgG4.pe analyses was within experimental error.
IgG1
In the human IgG1 assay, antibody C398.4A produced an IC50 of 1.2 ± 0.30nM for neutralization of human ICOS ligand and an IC50 of 0.14 ± 0.01nM for neutralization of mouse ICOS ligand.
IgG1 mAb STIM001, STIM002, STIM003 and STIM005 produced an IC50 similar to C398.4A using the human ICOS ligand neutralization system and where binding of mouse ICOS ligand to mouse ICOS receptor was also cross-reactive,
the other two cross-reactive mabs (STIM002-B and STIM004) showed weaker human and mouse ICOS ligand neutralization.
STIM006, STIM007, STIM008 and STIM009 showed neutralization of human ICOS ligand but did not exhibit significant cross-reactivity in the mouse ICOS ligand neutralization system. Neutralization IC50 values for mouse B7-H2 ligand may not be calculated against these antibodies.
| Average IC50(nM) | SD(nM)(n＝4) |
| STIM001 | 2.2 | 1.3 |
| STIM002 | 1.9 | 0.8 |
| STIM002-B | 3.6 | 3.5 |
| STIM003 | 1.3 | 0.5 |
| STIM004 | 233 | 123 |
| STIM005 | 2.5 | 0.8 |
| STIM006 | 2.2 | 1.5 |
| STIM007 | 1.1 | 0.5 |
| STIM008 | 1.6 | 1.4 |
| STIM009 | 30.5 | 53 |
| C398.4A | 1.2 | 0.3 |
Table E8-1 IC50 values for mabs of human IgG1 isotype that neutralize human ICOS receptor binding to human B7-H2. See also fig. 2.
| Average IC50(nM) | SD(nM)(n＝3) |
| STIM001 | 6.5 | 2.5 |
| STIM002 | 6.9 | 2.1 |
| STIM002-B | 30 | 11.4 |
| STIM003 | 0.1 | 0 |
| STIM004 | 22.1 | 15.4 |
| STIM005 | 0.3 | 0.2 |
| C398.4A | 0.1 | 0 |
Table E8-2 IC50 values for human IgG1 isotype mabs that neutralize binding of mouse ICOS receptor to mouse B7-H2. See also fig. 3.
IgG4.PE
As expected, IgG4.pe mAb gave similar results to IgG1 isotype.
STIM001, STIM003, and STIM005 showed IC50 values similar to C398.4A using the human ICOS ligand neutralization system. These mabs were also cross-reactive in neutralizing mouse ICOS ligands. STIM002-B and STIM004 produced weaker IC50 values against human ICOS B7-H2 neutralization and mouse B7-H2 ligand. STIM007, STIM008 and STIM009 showed neutralization of human ICOS ligand binding to human ICOS receptor, but the neutralization IC50 values of mouse B7-H2 ligand may not be calculated in these analyses.
The igg4.pe isoforms of STIM006 and STIM002 were not analyzed.
Table E8-3 IC50 values of human igg4.pe isotype mAb used to neutralize human ICOS receptor binding to human B7-H2. See also fig. 4.
| Average IC50(nM) | SD(nM)(n＝3) |
| STIM001 | 4.7 | 2.1 |
| STIM002-B | 43.9 | 25.7 |
| STIM003 | 0.2 | 0.1 |
| STIM004 | 30 | 14 |
| STIM005 | 0.3 | 0.1 |
| C398.4A | 0.2 | 0.1 |
Table E8-4 IC50 values of human igg4.pe isotype mabs used to neutralize mouse ICOS receptor binding to mouse B7-H2. See also fig. 5.
Materials and methods
The test antibody and isotype control were finally diluted in assay buffer (1 x PBS containing 0.53M potassium fluoride (KF), 0.1% Bovine Serum Albumin (BSA)) from starting working concentrations of up to 4 μ M, 1 μ M, to 0.002nM, 5.64e-4nM, finally by 11-point titration, 1/3 dilution. Mu.l of antibody was titrated into 384 well white-walled assay plates (Greiner Bio-One). Positive control wells received only 5. mu.l of assay buffer.
Mu.l of ICOS receptor (human ICOS-mFc, 20nM, finally 5nM or mouse ICOS-mFc, 4nM, finally 1nM (Chimerigen)) was added to the required wells. The plates were incubated at Room Temperature (RT) for 1 hour. After incubation, 5 μ l of mouse or human ICOS ligand conjugated to Alexa 647 (innovative biosciences) (B7-H2, addi bio) was diluted to 32nM (final 8nM) against human B7-H2 or 30nM, final 7.5nM against mouse B7-H2 and added to all wells of the assay plate except the negative control well that actually received 5 μ l of assay buffer.
Finally, 5 μ l of anti-mouse IgG donor mAb labeled with europium cryptate (CisBio), 40nM, finally 10nM (southern biotechnology) was added to each well and the assay was left to stand in the dark at RT and incubated for an additional 2 hours after incubation the assay was read on an Envision plate reader (perkin elmer) using standard HTRF methods, 620nM and 665nM channel values were exported to microsoft spreadsheet (microsoft) and △ -F% and% neutralization calculated, the titration curve and IC50 value [ M ] were plotted using graphpad (prism), the IC50 value was calculated by converting the data using the equation X ═ log (X), the data converted using a fitting algorithm, log (inhibitor) and reaction-variable slope (four parameters) were then fitted using nonlinear regression.
△ -F% calculation:
665/620nm ratio for ratiometric data reduction.
Negative control of signal-average of minimum signal ratio
And% neutralization:
example 9 a: t cell activation
STIM001 and STIM003 agonistic potential at cytokine production was tested in a culture plate-bound and soluble form in a human primary T cell activation assay when anti-CD 3 and anti-CD 28Ab were added in parallel to anti-ICOS Ab to induce ICOS expression on effector T cells. The effect of ICOS co-stimulation on IFN γ levels produced by these activated T cells was assessed 72 hours after activation using ELISA.
Materials and methods
T cell activation assay 1:
isolation of monocytes (PBMCs) from human peripheral blood:
white blood cell cones were collected from healthy donors and their contents were diluted up to 50mL with phosphate buffered saline (PBS from gepiroceae) and layered onto the top of 15mL Ficoll-Paque (from GE Healthcare) into 2 centrifuge tubes. PBMCs were separated by density gradient centrifugation (400g, 40 min, without brake), transferred to clean centrifuge tubes and then washed with 50mL PBS, twice with 300g centrifugation for 5 min and twice with 200g centrifugation for 5 min. PBMC were then resuspended in R10 medium (RPMI + 10% heat-inactivated fetal bovine serum, both from Gembidae) and plated with EVETMCells were counted and viability assessed in an automated cell counter (from NanoEnTek).
ICOS antibody (Ab) preparation and dilution:
STIM001 and STIM003 were tested in the following 3 formats: soluble plus F (Ab') of plate-bound form, soluble form or cross-linked anti-ICOS Ab2Fragment (109-006-170 from Jackson immunization study).
For the plate-bound form: anti-ICOS Ab and its isotype control 1:3 were serially diluted in PBS to obtain final antibody concentrations ranging from 45 μ g/mL to 0.19 μ g/mL. 100 μ L of diluted antibody was spread twice in duplicate at 4 ℃ onto 96-well highly-bound flat-bottomed plates (Corning EIA/RIA plates) overnight. The plates were then washed with PBS and 125 μ l of R10 was added to each well.
For the soluble form: anti-ICOS Ab and its isotype control 1:3 were serially diluted in R10 medium to give a2 × Ab stock concentration ranging from 90 μ g/mL to 0.38 μ g/mL. Two replicate aspirations of 125. mu.l of diluted Ab were pipetted into 96-well flat-bottom plates.
For the crosslinked soluble form: anti-ICOS Ab and isotype controls were compared to F (Ab')2The fragments were mixed in a ratio of 1M to 1M. Ab/F (Ab')2Fragment mixture 1:3 Serial dilutions in R10 medium to give a2 Xab concentration in the range of 90. mu.g/mL to 0.38. mu.g/mL for ICOS and F (Ab')2Fragments were at 2 Xab concentrations of 60. mu.g/mL to 0.24. mu.g/mL. Two replicate aspirations of 125. mu.l of diluted Ab were pipetted into 96-well flat-bottom plates.
T cell isolation, culture and IFN- γ quantification:
t cells were negatively isolated from PBMC using the EasySep human T cell isolation kit (from Stemcell Technologies) and processed at 2X 106The individual/mL were resuspended in R10 medium supplemented with 40. mu.l/mL of the dinosaur bead human T-activator CD3/CD28 (from Life technologies).
125. mu.l of T cell suspension was added to the Ab-containing plate to obtain 1X 106Final cell concentration per ml and at 37 ℃ and 5% CO2Next, it was cultured for 72 hours. Cell-free supernatants were then collected and kept at-20 ℃ until secreted IFN γ was analyzed by ELISA (duoset kit from addi bio).
This experiment was repeated on T cells isolated from 6 independent donors and each assay condition included 2 technical replicates.
T cell activation assay 2(STIM-REST-STIM assay):
the potential for STIM001 and STIM003 agonism on cytokine release was also tested in a plate binding assay in human T cell assays when T cells were pre-stimulated with anti-CD 3 and anti-CD 28Ab for 3 days prior to resting for 3 days to reduce the level of activation of T cells to induce ICOS expression, ICOS expression was confirmed by FACS staining after stimulation (day 3) and resting (day 6), these rested stimulated T cells were then cultured with STIM001 or STIM003 in the presence or absence of CD3Ab to assess TCR binding requirements, the effect of ICOS co-stimulation on the levels of IFN γ, TNF α and IL-2 present in the culture was assessed after 72 hours.
ICOS Ab dilution and coating:
anti-human CD3 (clone line UCHT1 from electronic bioscience) was diluted to a2 × Ab concentration of 10 μ g/mL in PBS. 50 μ l of PBS or 50 μ l of diluted CD3Ab were pipetted into 96-well highly-bound flat-bottom plates. STIM001, and its isotype control 1:2, were serially diluted in PBS to give 2 × antibody concentrations ranging from 20 μ g/mL to 0.62 μ g/mL. 50 μ L of diluted anti-ICOS Ab was added to wells containing PBS (no TCR binding) or diluted CD3Ab (TCR binding). The plates were plated overnight at 4 ℃.
T cell isolation, culture and IFN- γ quantification:
PBMCs from white blood cell cones were obtained as described in T cell activation assay 1. T cells were negatively isolated from this PBMC using EasySep human T cell isolation kit (from stem cell technology). T cells were cultured at 1X 106Each/mL was resuspended in R10 medium supplemented with 20. mu.l/mL of dinobead human T-activator CD3/CD28 (from Life technologies) and incubated at 37 ℃ and 5% CO2The cells were cultured for 3 days (stimulation). Dinotide beads were removed from the culture on day 3. The T cells were then washed (300g for 5 min), counted and counted at 1.5X 106Resuspended in R10 medium at 37 ℃ and 5% CO2The cells were cultured for another 3 days (resting stage).
On day 6, the T cells after resting on stimulation were then washed (300g for 5 min), counted and counted at 1X 106one/mL was resuspended in R10 media, and 250. mu.l of T cell suspension was added to ICOS Ab-coated plates and incubated at 37 ℃ and 5% CO2And culturing for 72 hours. The cell-free supernatant was then collected and maintained at-20 ℃ until the secreted cytokines were analyzed on the MSD platform.
This experiment was repeated with T cells isolated from 5 independent donors and each assay condition included 3 technical replicates.
Results
Two STIM001 and STIM003 tested positive for inducing IFN γ expression therefore showed agonism in both assays.
Example 9 b: t cell activation assay 1 data
Each STIM001 and STIM003 in the form of human IgG1 was tested using T cells isolated from 8 independent donors for T cell activation assay 1 as described in example 9 a. Hamster anti-ICOS antibody C398.4A and hamster antibody isotype controls were included for comparison. Each analysis condition included 2 technical replicates.
The results are shown in fig. 16, 17 and 18. As mentioned before, the two STIM001 and STIM003 tested positive for inducing IFN γ expression thus showed agonistic effects on human primary T cells.
Cross-linked antibodies act as agonists of T cell activation, as compared to soluble antibodies or to controls, such as in Fc-linked F (ab')2The strong enhancement induced by IFN γ in the presence of the fragment is indicated. IFN γ expression in T cells increased with increasing concentrations of cross-linked STIM001 or STIM003 (fig. 16, lower panel). Agonism was also observed for both STIM001 and STIM003 in plate-bound form, and for hamster antibody C398.4A was more weakly agonized as indicated by the observed increase in IFN γ expression in T cells with increasing antibody concentration (fig. 16, upper panel).
The magnitude of the IFN γ response varied between T cells obtained from different donors, but the expression of IFN γ by STIM001 increased continuously compared to that observed with the control antibody (HCIgG 1). When considering data from analyses performed with T cells from all 8 donors, it can be seen that treatment of T cells with STIM001 significantly increased IFN γ expression in the plate-bound, soluble and cross-linked forms compared to treatment with isotype control antibody (fig. 17). STIM001 thus appears to be an agonist of T cell activation in all three forms.
Similar effects were observed with STIM003 (fig. 18). The level of IFN γ induced by STIM003hIgG1 was compared to that induced by its isotype control (HC IgG1) at the given dose of antibody in the assay against 8 independent donors. The average increase in IFN γ levels induced by STIM003 was significant compared to HC IgG1, regardless of donor-to-donor variation. STIM001 and other STIM antibodies described herein are proposed to have the potential to similarly promote T cell activation in vivo. As previously discussed, agonism of activated ICOS-expressing T cells may be mediated by anti-ICOS antibodies that bind to ICOS receptors on the surface of T cells and induce multimerization thereof. Example 9 c: t cell activation assay 2 data
T cell activation assay 2 was performed as described in example 9 a.
In the absence of TCR binding (no anti-CD 3 antibody), the levels of cytokines produced by primary T cells were lower and were not increased even at the highest concentration of 10 μ g/ml induced by STIM001(hIgG1), STIM003(hIgG1) or antibody C398.4A in contrast, when anti-ICOS antibody was added to T cells in combination with anti-CD 3 antibody, each of STIM001(hIgG1), STIM003(hIgG1) and C398.4A showed a dose-dependent trend to increase expression of IFN γ, TNF α and to a lesser extent IL-2.
Data for treatment of primary T cells with anti-ICOS antibody under TCR binding conditions is shown in fig. 19. Although a significant increase in cytokine expression was observed for each of STIM001, STIM003, and C389.4A relative to its corresponding isotype control, the differences did not reach statistical significance in this analysis. Further analysis repetitions with reactive primary T cells from more donors would be expected to yield statistically significant results.
Example 10 a: ADCC assay
The potential of STIM001 and STIM003 for killing by ADCC was tested in a Delfia wada cytotoxicity assay (perkin elmer) using human primary NK cells as effectors and an ICOS high MJ cell line (ATCC, CRL-8294) as target cells. MJ cells are human CD 4T lymphocytes expressing high levels of ICOS protein.
This method is based on loading target cells with a fluorescence enhancing ligand acetoxymethyl ester (BATDA) that rapidly penetrates the cell membrane. Within the cell, the ester bond is hydrolyzed to form a hydrophilic ligand (TDA) that no longer crosses the membrane. After cell lysis, the ligand is released and can be detected by the addition of europium, which forms a highly fluorescent and stable chelate (EuTDA) with BATDA. The measured signal is directly related to the amount of lysed cells.
Materials and methods
Labeling of target cells:
MJ cells were plated at 1X 10 according to the manufacturer's instructions6Each/mL was resuspended in assay medium (RPMI + 10% ultra-low IgG FBS, from Gidbaceae) and loaded with 5. mu.l/mL of Delfia BATDA reagent (Perkin Elmer) for 30 minutes at 37 ℃. MJ cells were then washed 3 times with 50mL PBS (300g, 5 min) and at 8X 105Each ml was resuspended in assay medium supplemented with 2mM probenecid (from life technologies) to reduce spontaneous release of BATDA from the cells.
ICOS Ab dilution:
STIM001, STIM003, and their isotype controls 1:4 were serially diluted in assay medium +2mM probenecid to give a final 4 x antibody concentration in the range down to 80 pg/mL.
NK cell isolation and culture:
PBMCs from white blood cell cones were obtained as described in T cell activation assay 1. NK cells were negatively isolated from this PBMC using the EasySep human NK cell isolation kit (from Stem cell technology) and at 4X 106Each/ml was resuspended in R10 medium +2mM probenecid. NK cell purity was greater than 90% as checked by staining CD3-/CD56 +.
To each well was added 50 μ l of diluted Ab, 50 μ l of BATDA loaded with MJ cells, 50 μ l of NK cells and 50 μ l of assay medium +2mM probenecid (final volume 200 μ l/well) to give a final Ab concentration in the range down to 20pg/mL and an effector to target ratio of 5: 1. Wells containing MJ cells only or MJ cells + delfia lysis buffer (perkin elmer) were used to determine spontaneous release and 100% bat da release.
50 μ l of cell-free supernatant was transferred to DELFIA microtiter plates (Perkin Elmer) at 37 deg.C with 5% CO2The analysis was performed for 2 hours. 200 μ l of Delfia europium solution (Perkin Elmer) was added to the supernatant and incubated at room temperature for 15 min. The fluorescent signal was then quantified using an Envision multi-label reader (perkin elmer).
Specific release induced by STIM001 and STIM003 were calculated according to the kit instructions. This experiment was repeated with NK cells from independent donors and each assay condition included 3 technical replicates.
Results
In a primary NK-dependent ADCC assay (2 hour time point), anti-ICOS antibodies STIM001(hIgG1) and STIM003(hIgG1) killed ICOS-positive human MJ cells. See also fig. 6 a. Sub-nanomolar EC50 was obtained for the two tested molecules in this analysis.
| EC50 | Donor 1 | Donor 2 |
| STIM001 | 1.21e-10 | 5.29e-10 |
| | |
Table E10-1: EC50 (molar units) of STIM001 in NK primary cell ADCC assay (2 hour time point) from 2 donors.
Example 10 b: ADCC assay with MJ target cells
Experiments were performed according to the materials and methods set forth in example 10 a. STIM001, STIM003, and isotype control 1:4 were serially diluted in assay medium +2mM probenecid to give a final 4 × antibody concentration in the range of 40 μ g/mL to 80 pg/mL. Mu.l of diluted Ab, 50. mu.l of BATDA loaded with MJ cells, 50. mu.l of NK cells and 50. mu.l of assay medium +2mM probenecid (final volume 200. mu.l/well) were added to each well to give a final Ab concentration in the range of 10. mu.g/mL to 20pg/mL and an effector to target ratio of 5: 1.
The results are shown in fig. 6(6 b-6 d) and the table below. STIM001(hIgG1) and STIM003(hIgG1) killed ICOS-positive human MJ cells in a primary NK-dependent ADCC assay (measured at the two hour time point).
| EC50 | Donor 1 | Donor 2 | Donor 3 |
| STIM001 | 1.21e-10(0.121nM) | 5.29e-10(0.529nM) | 2.92e-09(2.92nM) |
| STIM003 | 2.33e-12(2.33pM) | 3.58-e-11(35.8pM) | 1.01e-10(0.101nM) |
Table E10-2: EC50 (molar units) for STIM001 and STIM003 in NK primary cell ADCC assays (2 hour time point) from 3 donors.
Example 10 c: ADCC assay with ICOS transfected CCRF-CEM target cells
The potential of STIM001 and STIM003hIgG1 for killing by ADCC was further tested in a Delfia wada cytotoxicity assay (perkin elmer) using human primary NK cells as effectors and ICOS-transfected CCRF-CEM cells (ATCC, CRL-119) as target cells. CCRF-CEM is a human T lymphoblastic cell line derived from peripheral blood in patients with acute lymphoblastic leukemia. Antibody-mediated killing of CCRF-CEM cells was confirmed for both STIM001 and STIM003 in this assay.
Materials and methods
The materials and methods were as set forth in example 10a, but CCRF-CEM cells obtained from ATCC (ATCC CCL-119) were used as target cells instead of MJ cells, and an incubation time of 4 hours was used.
CCRF-CEM cells were transfected with ICOS. Synthetic string DNA encoding full-length human ICOS (with a signal peptide, as shown in the accompanying sequence listing), which was codon optimized for mammalian expression, was cloned into an expression vector under the control of the CMV promoter and flanked by 3 'and 5' piggyBac-specific terminal repeats to facilitate stable integration into the cell genome (see [40 ]). The expression vector contains a puromycin selection cassette to facilitate stable cell line production. Human ICOS expression plasmids were co-transfected with plasmids encoding piggyBac transposase into CEM CCRF cells by electroporation. 24 hours post transfection, media was supplemented with puromycin and grown for at least two weeks to select stable cell lines, with media changed every 3-4 days. Expression of human ICOS was assessed by flow cytometry using anti-human ICOS-PE conjugated antibodies (electron biosciences). The complete CEM medium consisted of higher RPMI medium containing 10% (v/v) FBS and 2mM Glutamax.
STIM001(hIgG1), STIM003(hIgG1) and isotype control antibody (HC IgG1) were serially diluted in assay medium to give a final 4 x antibody concentration in the range of 20 μ g/mL to 80 pg/mL.
To each well was added 50. mu.l of diluted Ab, 50. mu.l of BATDA loaded with ICOS transfected CEM cells, 50. mu.l of NK cells and 50. mu.l of assay medium (final volume of 200. mu.l/well) to give a final Ab concentration in the range of 5. mu.g/mL to 20pg/mL and an effector to target ratio of 5: 1.
Results
In a primary NK-dependent ADCC assay (measured at the four hour time point), STIM001(hIgG1) and STIM003(hIgG1) killed ICOS-transfected CCRF-CEM cells. The results are shown in fig. 6(6 e-6 g) and in the table below.
| EC50 | Donor 4 | Donor 5 | Donor 6 |
| STIM001 | 3.92e-12(3.92pM) | 3.95e-12(3.95pM) | 3.75e-12(3.75pM) |
| STIM003 | Approximately 3pM | 8.95e-13(0.895pM) | 1.03e-12(1.03pM) |
Values estimated from incomplete curves.
Table E10-3: EC50 (molar units) for STIM001 and STIM003 in NK primary cell ADCC assays (4 hour time point) from 3 donors.
Example 11 a: CT26 isogenic model
Improved in vivo efficacy against tumors by combining anti-ICOS (STIM001 mIgG2a, effector priming) with anti-PDL 1(10F9G2) was shown in the CT26 isogenic model.
Materials and methods
Efficacy studies were performed in Balb/c mice using the subcutaneous CT26 colon cancer model (ATCC, CRL-2638). This model has lower sensitivity to PD1/PDL1 blockade, and tumor growth-only retardation (unstable disease or cure) in response to 10f9.g2 (anti-PDL 1) and RMT1-14 (anti-PD 1) monotherapy is often observed. Thus, this model constitutes a relevant model for looking at the intrinsic resistance of anti-PD 1, anti-PDL 1 for combinatorial studies. All in vivo experiments were performed under the UK ministry of medicine Project license (UK Home Office Project license) according to the UK Animal (Scientific Procedures) Act 1986 and EU Directive 86/609 and approved by the babaham Institute of Animal Welfare (babaham Institute Animal Welfare) and the Ethical Review agency Ethical Review Body.
Supplied by Charles River UK for 6 to 8 weeks of age and>18g of Balb/c mice were housed in a pathogen-free environment. Will be 1X 10 in total5Individual CT26 cells (passage number below P20) were injected subcutaneously into the right flank of the mice. Unless otherwise stated, noTreatment was started on day 6 after tumor cell injection. CT26 cells were passaged ex vivo by using cell digest (sigma), washed twice in PBS and resuspended in RPMI supplemented with 10% fetal bovine serum. Cell viability was confirmed to be above 90% at tumor cell injection.
For in vivo studies, STIM001 anti-ICOS agonists (cross-reactive to mouse ICOS protein) were reformatted as mouse IgG 1and mouse IgG2a to be tested when effector function was ineffective and when effector function was enabled, respectively. anti-PDL 1 was obtained from Association Biotech Inc. (Biolegend) (Cat. No.: 124325). Hybridization controls were generated in Kymab (mIgG2a isotype) or from commercial sources (hamster isotype HTK888, associative biotechnology limited (product No. 92257, batch B215504)). All antibodies were administered Intraperitoneally (IP) three times a week starting on day 6 (2 weeks, 6 to 18 days) at 10mg/kg (1mg/ml in 0.9% normal saline) as monotherapy or by combining anti-PDL 1 with anti-ICOS antibody. Animal weight and tumor volume were measured 3 times per week starting on the day of tumor cell injection. Tumor volume was calculated by using the modified ellipsoid formula 1/2 (length x width 2). Mice were studied continuously until mice tumors reached 12mm3Until or in rare cases when tumor ulceration is observed (welfare). The experiment was stopped on day 50. Human endpoint survival statistics were calculated by Prism using the Kaplan-Meier method (Kaplan-Meier method). This method was used to determine whether specific treatment was associated with improved survival.
Table E11-1: treatment group
Results
As shown in fig. 7, 8 and 9, ICOS agonists may delay disease progression and cure CT-26 subcutaneous tumors in a fraction of animals as monotherapy or in combination with anti-PDL 1. anti-PDL 1 monotherapy induced a delay in tumor growth but no stable disease or curative potential was observed. The combination is more effective than anti-ICOS monotherapy in treating tumors. The present study also highlights that STIM001 in the form of mouse IgG2a (effector function enabled) was more effective than in the form of mouse IgG1 (effector null) in eliciting an anti-tumor response in this model.
Example 11 b: stronger activity of the combination of anti-ICOS mIgG2a and anti-PDL 1mIgG2a in the CT26 isogenic modelIn vivo antitumor efficacy
In vivo combination studies were performed with AbW designated with STIM001 cross-reactive anti-human PDL1 antibody. With respect to this in vivo effect, STIM001 was reformatted into mouse IgG 1and mouse IgG2a to compare its efficacy to low effector function or to compare its efficacy as an effector function-enabling molecule, respectively. anti-PDL 1 AbW was produced in the same format (mouse IgG 1and mouse IgG2 a).
Efficacy studies were performed in Balb/c mice using the subcutaneous CT26 colon cancer model (ATCC, CRL-2638). Supplied by Charles River UK for 6 to 8 weeks of age and>18g of Balb/c mice were housed in a pathogen-free environment. A total of 1 × 10E5CT26 cells (passage number below P20) were injected subcutaneously into the right flank of the mice. Unless otherwise stated, treatment was initiated on day 6 post tumor cell injection. CT26 cells were obtained by using trypLETMThe expressed enzyme (semer feisher) was passaged in vitro, washed twice in PBS and resuspended in RPMI supplemented with 10% fetal bovine serum. Cell viability was confirmed to be above 90% at tumor cell injection.
200 μ g each of STIM001 and anti-PDL 1 antibody (1mg/ml in 0.9% physiological saline) were administered in combination simultaneously Intraperitoneally (IP) three times a week from day 6 after tumor cell implantation (2 weeks between day 6 and day 17). Tumor growth was monitored and compared to control antibodies with isotype (mIgG 1and mIgG 2)A) The mixture of (a) is compared to a tumor in an animal treated with the mixture of (b). Animal weight and tumor volume were measured 3 times a week starting on the day of tumor cell injection. Tumor volume was calculated by using the modified ellipsoid formula 1/2 (length x width 2). Mice were studied continuously until mice tumors reached 12mm3By the mean diameter of (a) or in rare cases when tumoral ulceration is observed (welfare). The experiment was stopped on day 60. Human endpoint survival statistics were calculated by Prism using the Kaplan-Meier method (Kaplan-Meier method). This method was used to determine whether specific treatment was associated with improved survival.
| Group of | Number of animals | Treatment regimen (3 times a week for 2 weeks) |
| 1 | 10 | mIgG2a + mIgG1 isotypes each 200 μ g |
| 2 | 10 | anti-ICOS mIgG1 STIM001+ anti-PD-L1 mIGg1(AbW) each 200. mu.g |
| 3 | 10 | anti-ICOS mIgG2a STIM001+ anti-PD-L1 mIGg2a (AbW) each 200. mu.g |
| 4 | 10 | anti-ICOS mIgG2a STIM001+ anti-PD-L1mIGg1(AbW) each 200. mu.g |
| 5 | 10 | anti-ICOS mIgG1 STIM001+ anti-PD-L1 mIGg2a (AbW) each 200. mu.g |
Table E11-2: treatment group of STIM 0012 x 2 combinations
The results are shown in fig. 10. All antibody combinations delay tumor growth and prolong the survival (time to human endpoint) of treated animals when compared to isotype control treated animals. Interestingly, STIM001 mIgG2a antibody was more effective in inhibiting tumor growth than STIM001 in the mIgG1 form when combined with anti-PDL 1 (mIgG1 or mIgG2a, regardless of its form). These data indicate that anti-ICOS antibodies have the advantage of effector function to maximize anti-tumor efficacy. Notably, STIM001 mIgG2a in combination with aPD-L1 mIgG2a has demonstrated the strongest antitumor efficacy and improved survival (90% of the animals showed a response and 60% were cured from disease at day 60).
Similarly, STIM003mIgG 1and mIgG2a were tested as monotherapies or in combination with anti-PDL 1(AbW) mIgG2a in the same CT26 tumor model. Starting on day 6 after tumor cell implantation, STIM003 and anti-PDL 1 antibodies were administered to the animals each 200 μ g of antibody (1mg/ml in 0.9% physiological saline) as monotherapy or in combination by intraperitoneal Injection (IP) three times a week (2 weeks between days 6 and 17). In this experiment, tumor size was monitored for 41 days. Human endpoint survival statistics were calculated by Prism using the Kaplan-Meier method (Kaplan-Meier method). This method was used to determine whether specific treatment was associated with improved survival.
| Group of | Number of animals | Treatment regimen (3 times a week for 2 weeks starting on day 6) |
| 1 | 10 | mIgG2a + mIgG1 isotype controls each 200 μ g |
| 2 | 10 | anti-PD-L1 mIgG2a (AbW) 200. mu.g |
| 3 | 10 | STIM003 mIgG1 200μg |
| 4 | 10 | STIM003 mIgG2a 200μg |
| 5 | 10 | STIM003mIgG 1+ anti-PD-L1 mIGg2a (AbW) each 200. mu.g |
| 6 | 10 | STIM003mIgG2a + anti-PD-L1 mIGg2a (AbW) each 200. mu.g |
Table E11-3: treatment group combining STIM003 with anti-PDL 1 AbW IgG2a
The results are shown in fig. 11. Monotherapy with pdl1(AbW) and STIM003mIgG2a has demonstrated mild anti-tumor activity (cure of disease in one animal per group). STIM003mIgG 1 or mIgG2a in combination with pdl1(AbW) mIgG2a have shown greater anti-tumor efficacy. Interestingly, by day 41, STIM003mIgG2a was more effective in inhibiting tumor growth than STIM003mIgG 1 when combined with pdl 1mIgG2a (60% cured disease in 30% of animals, respectively). The data further highlight the advantage of the effector forms of anti-ICOS antibodies to maximize anti-tumor efficacy.
Taken together, these data demonstrate that the combination of anti-ICOS antibody STIM001 or STIM003 with anti-PDL 1 produced the strongest anti-tumor response when both antibodies had effector-activating function. Suitable corresponding human antibody isotypes will include human IgG1 optionally with further enhanced effector function, e.g., afucosylated IgG 1.
Mice treated with the combination of anti-PDL 1mIgG2a and STIM003mIgG2a and kaplan meier curves treated with each agent alone are shown in figure 29.
Example 11 c: single dose of STIM003 antibody in combination with sustained anti-PD-L1 administration resets the tumor microenvironment(TME) and produces a strong antitumor effect
The study compared single and multiple doses of STIM003mIgG2A and multiple doses of anti-PDL 1 antibody (AbW). The data indicate that a single dose of anti-ICOS antibody can alter the tumor microenvironment in order to allow the anti-PD-L1 antibody to exert greater effects. This situation can be envisaged as "resetting" the TME by the anti-ICOS antibody.
These efficacy studies were performed in Balb/c mice using the subcutaneous CT26 colon cancer model (ATCC, CRL-2638) as previously described. Supplied by Charles River UK for 6 to 8 weeks of age and>18g of Balb/c mice were housed in a pathogen-free environment. A total of 1 × 10E5CT26 cells (passage number below P20) were injected subcutaneously into the right flank of the mice. Unless otherwise stated, treatment was initiated on day 6 post tumor cell injection. CT26 cells were obtained by using trypLETMThe expressed enzyme (Saimerfield) was passaged ex vivo and washed in PBSWashed twice and resuspended in RPMI supplemented with 10% fetal bovine serum. Cell viability was confirmed to be above 90% at tumor cell injection.
Treatment groups are shown in table E11-4. STIM003 and anti-PDL 1 antibody were administered Intraperitoneally (IP) at 10mg/kg (1mg/mL in 0.9% physiological saline). Treatment started on day 6 after tumor cell implantation. Tumor growth was monitored and compared to tumors in animals treated with saline. Animal weight and tumor volume were measured 3 times a week starting on the day of tumor cell injection. Tumor volume was calculated by using the modified ellipsoid formula 1/2 (length x width 2). Mice were studied continuously until mice tumors reached 12mm3By the mean diameter of (a) or in rare cases when tumoral ulceration is observed (welfare). The experiment was stopped on day 55.
The data is shown in figure 34. Concurrent STIM003 and anti-PDL 16 doses from day 6 gave greater anti-tumor efficacy in the CT26 model, with 5/8 animals having no tumor at the end of the study (day 55). Interestingly, similar anti-tumor efficacy has been achieved with a single dose of STIM003 followed by multiple doses of anti-PDL 1 as monotherapy. Similar overall efficacy was observed between administration of STIM003 once (C) versus 6 times (B) when combined with anti-PD-L1 mIgG2 a. The group treated with the combination drug had complete tumor rejection in 62.5% of the animals by the end of the experiment (day 55) when compared with the saline-treated group (a) in which only one animal had spontaneous tumor rejection (rare in this model). The data indicate that STIM003 antibodies can be used to re-establish the tumor microenvironment, and that the antibodies allow immune checkpoint resistant tumors to become sensitive against PDL 1. As previously demonstrated (example 11b), the CT26 tumor cell line did not respond strongly to anti-PDL 1 monotherapy. STIM003 was shown to produce changes in antitumor activity that promote anti-PDL 1 therapy.
Example 12: antibody sequence analysis
The framework regions of antibodies STIM001, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009 were compared to the human germline gene segments to identify the closest match. See Table E12-1 and Table E12-2.
| Heavy chain | V | D | J |
| STIM001 | IGHV1-18*01 | IGHD6-19*01 | IGHJ6*02 |
| STIM002 | IGHV1-18*01 | IGHD3-10*01 | IGHJ6*02 |
| STIM002-B | IGHV1-18*01 | IGHD3-10*01 | IGHJ6*02 |
| STIM003 | IGHV3-20*d01 | IGHD3-10*01 | IGHJ4*02 |
| STIM004 | IGHV3-20*d01 | IGHD3-10*01 | IGHJ4*02 |
| STIM005 | IGHV1-18*01 | IGHD3-9*01 | IGHJ3*02 |
| STIM006 | IGHV3-11*01 | IGHD3-10*01 | IGHJ6*02 |
| STIM007 | IGHV2-5*10 | IGHD3-10*01 | IGHJ6*02 |
| STIM008 | IGHV2-5*10 | IGHD3-10*01 | IGHJ6*02 |
| STIM009 | IGHV3-11*01 | IGHD3-9*01 | IGHJ6*02 |
TABLE E12-1 heavy chain germline Gene segments of anti-ICOS Ab
| Light chain | V | J |
| STIM001 | IGKV2-28*01 | IGKJ4*01 |
| STIM002 | IGKV2-28*01 | IGKJ2*04 |
| STIM002-B | IGKV2-28*01 | IGKJ2*04 |
| STIM003 | IGKV3-20*01 | IGKJ3*01 |
| STIM004 | IGKV3-20*01 | IGKJ3*01 |
| STIM005 | IGKV1D-39*01 | IGKJ1*01 |
| STIM006 | IGKV2-28*01 | IGKJ2*04 |
| STIM007 | IGKV3-11*01 | IGKJ4*01 |
| STIM008 | IGKV3-11*01 | IGKJ4*01 |
| STIM009 | IGKV2-28*01 | IGKJ1*01 |
TABLE E12-2 kappa light chain germline Gene segments of anti-ICOS Ab
Additional antibody sequences were obtained by next generation sequencing of PCR amplified antibody DNA from other ICOS-specific cells sorted from immunized mice as described in example 3. This identified a large number of antibodies that could be grouped into clusters with STIM001, STIM002 or STIM003 based on their heavy and light chain j gene segments and CDR3 lengths. CL-61091 is clustered with STIM 001; CL-64536, CL-64837, CL-64841 and CL-64912 cluster with STIM 002; and CL-71642 and CL-74570 cluster with STIM 003. A sequence alignment of the antibody VH and VL domains is shown in figures 35 to 37.
Table E12-3 antibodies clustered by sequence.
Example 13: agonism of ICOS-expressing MJ cells by bead-bound antibodies
Antibodies STIM001, STIM002 and STIM003, anti-ICOS antibody C398.4A and ICOS ligand (ICOSL-Fc) were each covalently coupled to beads and evaluated for their ability to induce expression of cytokine IFN- γ in MJ cells grown from culture. Human IgG1 coupled to beads and a clone C398.4A isotype control were evaluated simultaneously.
The data is shown in fig. 12 and table E13 below.
Each of the anti-ICOS antibodies had exhibited agonism in this assay, stimulating MJ cells significantly over the dynamic range of the assay as determined by IFN- γ quantification, than observed by its cognate isotype control.
STIM003 and clone C398.4A gave lower top asymptotic values (95% CI: 3.79 to 5.13 and 3.07 to 4.22, respectively) but more effective LogEC50 values (95% CI: 9.40 to-9.11 and-9.56 to-9.23, respectively) compared to STIM001 (top 95% CI: 7.21 to 8.88 and LogEC 5095% CI: -8.82 to-8.63) and STIM002 (top 95% CI: 5.38 to 6.95 and LogEC 5095% CI: -9.00 to-8.74). Because ICOSL-Fc and clone C398.4A isotype control produced incomplete curves (top outside the dynamic range of the analysis), the fitted top and LogEC50 values were not considered reliable. The human IgG1 hybridization control produced a complete curve, however, the area under the curve differed insignificantly from 0 and was therefore not considered an agonist.
Table e13 summary of bead-bound MJ cell activation assays in vitro. NA- -not applicable.
MJ cell activation assay materials and methods- -bead binding
Coupling of target proteins to magnetic particles
anti-ICOS antibody, control antibody and ICOSL-Fc were coupled to beads as follows.
M-450 tosyl activated (Tosylactivated) dino beads (Invitrogen; approximately 2X 10)8Beads/sample) were incubated with 100 μ g of each protein sample overnight at room temperature with stirring. The beads were washed three times with DPBS (Gilbecco) and incubated with 1M Tris-HCI (Ultrapure) at pH 8.0TMGembidae) were incubated together at room temperature under stirring for 1 hour to block non-coupling reactive sites. The beads were again washed three times with DPBS and finally resuspended in 0.5ml DPBS/sample.
The amount of each protein of interest on the beads was then determined as follows. Black flat bottom high binding ELISA plates (Gerania) were coated with DPBS containing 4. mu.g/ml of capture antibody against human IgG (southern Biotechnology) or hamster IgG (Jackson Immunity research), 50. mu.l/well overnight at 4 ℃. Each well was then washed three times with DPBS + 0.1% Tween, 200 μ l/well and blocked with 200 μ l/well DPBS + 1% BSA for 1 hour at room temperature. Each well was washed three more times with DPBS + 0.1% Tween. The stock protein samples were quantified by spectrometry and beads were counted using a cell counter. The dilution series of protein samples and beads were then incubated in the plate at 50 microliters/well for 1 hour at room temperature before being washed three more times with DPBS + 0.1% Tween. 50 μ l/well of DPBS + 0.1% BSA with biotinylated anti-hamster antibody or anti-human IgG-europium were added and incubated at room temperature for 1 hour. With the addition of biotinylated anti-submenu hamster antibody C398.4A, another incubation step added 50 μ l/well europium streptavidin (perkin elmer) diluted 1:500 in assay buffer (perkin elmer) and incubated for 1 hour at room temperature. Before developing the assay by adding 50 microliters/well of Delfia enhancing solution (perkin elmer), incubating for 10min at room temperature and measuring the emitted fluorescence at 615nm on an EnVision multi-label plate reader, each well was washed three times with 200 microliters/well of TBS + 0.1% Tween. The amount of protein on the beads was determined by extrapolating the value of the signal obtained from a sample of unconjugated protein of known concentration.
MJ cell in vitro activation assay- -bead binding
MJ[G11]The cell line (ATCC CRL-8294) was grown in IMDM (Gibbs or ATCC) supplemented with 20% heat-inactivated FBS. Cells were counted and 15000 cells/well (50 μ l/well) of the cell suspension was added to 96-well clear flat-bottom polystyrene sterile TC treated microplates. The beads were counted and ranged between 1.5X 106Addition of successive 1:2 dilutions of one bead/well to approximately 5860 beads/well (50 microliters/well) to the cells was repeated two or three times. To account for background, several wells of the plate contained only MJ cells (100 μ l/well). Cells and beads inIn plates at 37 ℃ and 5% CO2The following co-culture was performed for 3 days, after which the supernatant was collected by centrifugation and collected for determination of IFN-. gamma.content.
Measurement of IFN-gamma content
The IFN-. gamma.content in each well was determined using a modification of the human IFN-. gamma.DuoSet ELISA kit (Andy Bio Inc.). Capture antibody (50 μ l/well) was coated in DPBS at 4 μ g/ml onto a black flat bottom higher binding plate (geliana) overnight. Each well was washed three times with 200 μ l/well DPBS + 0.1% Tween. Each well was blocked with 200 μ l/well of 1% BSA in DPBS (w/v), washed three times with 200 μ l/well DPBS + 0.1% Tween, and then 50 μ l/well of RPMI or pure cell supernatant containing IFN- γ standard solution was added to each well and incubated at room temperature for 1 hour. Each well was washed three times with 200 μ l/well DPBS + 0.1% Tween before adding 50 μ l/well DPBS + 0.1% BSA containing 200ng/ml biotinylated detection antibody and incubating at room temperature for 1 hour. Each well was washed three times with 200 μ l/well DPBS + 0.1% Tween before adding 50 μ l/well of 1:500 europium streptavidin (perkin elmer) diluted in assay buffer (perkin elmer) and incubated for 1 hour at room temperature. Before the analysis was developed by adding 50 μ l/well of Delfia enhancing solution (perkin elmer) and incubating for 10min at room temperature and measuring the emitted fluorescence at 615nm on an EnVision multi-label plate reader, each well was washed three times with 200 μ l/well of TBS + 0.1% Tween.
Data analysis
The IFN- γ value for each well was interpolated from the standard curve and the mean background content from the cell wells alone was subtracted. The background correction values were then used in GraphPad prism to fit a 4-parameter log-log concentration response curve.
Example 14: agonism of ICOS-expressing MJ cells by plate-bound antibodies
An alternative assay for agonist effect of ICOS expressing T cells uses antibodies in a plate-bound form.
MJ cell activation assay materials and methods- -plate binding
Antibody coating: 96-well sterile flat higher binding plates (Costar) were prepared at 4 ℃ using 100 μ l/well of serial 1:2 dilutions (ranging from 10. mu.g/ml to 0.02. mu.g/ml) were repeated twice or three times overnight. Considering background, several wells of the culture plate were coated with DPBS only. The plates were then washed three times with 200 μ l/well DPBS before adding the cells.
Cell stimulation: MJ [ G11]The cell line (ATCC CRL-8294) was grown in IMDM (Gibbs or ATCC) supplemented with 20% heat-inactivated FBS. Cells were counted and a cell suspension of 15000 cells/well (100 microliters/well) was added to the protein-coated culture plate. Cells were cultured in plates at 37 ℃ and 5% CO2The cells were cultured for 3 days. Cells were separated from the medium by centrifugation and the supernatant was collected for determination of IFN- γ content.
Measurement and data analysis of IFN γ content were as described in example 13.
Results
The results are shown in FIG. 13 and Table E14-1 below. Taken together, STIM001, STIM002 and STIM003 all showed potent agonism as measured by IFN- γ secretion, with similar LogEC50 values (LogEC 5095% CI: -7.76 to-7.64, -7.79 to-7.70 and-7.82 to-7.73, respectively) and top values (top 95% CI: 2.06 to 2.54, 2.44 to 2.93 and 2.01 to 2.41, respectively). Clone C398.4A exhibited similar LogEC50 values (LogEC 5095% CI: -7.78 to-7.60) but lower top values (top 95% CI: 1.22 to 1.63) as STIM001 to STIM 003. STIM004 also showed agonism in this assay, but the effect was lower, with moderate top values (top 95% CI: 0.16 to 0.82) and similar LogEC50 values (LogEC 5095% CI: -7.91 to-7.21) being achieved. STIM001, STIM002 and STIM003 are stronger agonists than ICOSL-Fc (LogEC 5095% CI: -7.85 to-7.31 and top 95% CI: 0.87 to 2.45).
Table E14-1 summary of plate-bound MJ cell activation assays in vitro. IgG1 ═ human IgG1 hybridization control antibody.
Example 15: agonism of ICOS expressing MJ cells by antibodies in soluble form
In contrast to the assays described in examples 13 and 14 using antibodies arrayed on a solid surface, this assay determines whether the antibodies in soluble form act as agonists for ICOS-expressing T cells.
MJ cell activation assay materials and methods-soluble
MJ[G11]The cell line (ATCC CRL-8294) was grown in IMDM (Gibbs or ATCC) supplemented with 20% heat-inactivated FBS. Cells were counted and 15000 cells/well (50 μ l/well) of the cell suspension was added to a 96-well clear flat-bottom polystyrene sterile TC treated microplate. The addition of the target protein ranging from 10. mu.g/ml to 0.01953125. mu.g/ml was repeated twice or three times (50. mu.l/well) to the cells alone or in serial 1:2 dilutions with the addition of a cross-linker (Affinipure F (ab')2 fragment goat anti-human IgG, Fc fragment specificity; Jackson immunization study). To account for background, several wells of the plate contained only MJ cells (100 μ l/well). Cells and beads in culture plates at 37 ℃ and 5% CO2The following co-culture was performed for 3 days, after which the supernatant was collected by centrifugation and collected for determination of IFN-. gamma.content.
Measurement and data analysis of IFN γ content were as described in example 13.
Results
Both STIM001 and STIM002 showed significant soluble agonism as measured by IFN- γ secretion compared to human igg4.pe hybrid controls. Crosslinking of the mAb via goat anti-human IgG Fc F (ab')2 fragments even more increased the secreted IFN-. gamma.content.
Example 16: binding of antibodies to activated T cells
A. Human ICOS
The ability of anti-ICOS antibodies to recognize the ICOS extracellular domain on the surface of activated primary human T cells in their native state was confirmed in this assay.
Pan T cells (CD3 cells) were isolated and cultured with CD3/CD28 dino beads (sequofemtoler) for 3 days to induce ICOS expression on their surface. Surface staining of STIM001, STIM003 and hIgG1 hybridization controls (HC IgG1) was determined by two methods, namely detection after direct binding to a pre-labeled antibody (antibody conjugated directly to AF 647) or indirectly via the use of a secondary AF647 goat anti-human Fc antibody. Stained cells were run on Atttune and staining intensity was presented as the Mean of fluorescence intensity (Mean of fluorescence intensity; MFI). EC50 was determined using GraphPad Prism.
The results are shown in fig. 14. Once activated, pan CD3T cells were clearly stained by both STIM001 and STIM003hIgG 1. Notably, saturation of STIM003 binding to activated T cells occurred at lower concentrations than STIM001, indicating a higher affinity of STIM003 for human ICOS. EC50 for STIM003 was roughly 100-fold lower than EC50 for STIM001 (0.148 nM vs 17nM for indirect binding assays).
B. ICOS from non-human primates
The ability of anti-ICOS antibodies to recognize, in their native state, the ICOS extracellular domain on the surface of activated primary T cells from non-human primates (NHPs) was confirmed in this assay.
PBMCs from whole blood of 2 Machiki monkeys (Wickham Laboratories) were isolated by gradient centrifugation and cultured for 3 days with CD2/CD3/CD28 MACSiBeads (Miltenyi) to induce ICOS expression on their surface. Surface staining of the STIM001, STIM003 and hIgG1 hybridization controls (HC IgG1) was determined after direct binding of AF647 pre-labeled antibody (80. mu.g to 8 pg/ml). Cells were also labeled with V450-CD3 to assess staining on T cell subsets. Stained cells were run on Attune (semer fematile) and staining intensity was presented as Mean Fluorescence Intensity (MFI). EC50 was determined using GraphPadPrism.
The results are shown in fig. 28. Once activated, T cells were clearly stained by both STIM001 and STIM003hIgG 1. As observed with binding to human T cells, saturation of STIM003 binding to activated NHP T cells occurred at lower concentrations than STIM001, indicating STIM0003 has higher affinity in both antibodies ICOS. EC50 values associated with NHP ICOS were similar to those obtained in association with human ICOS.
TABLE E16 calculation of EC50 (Mole) for antibodies binding to ICOS on activated NHP T cells
Example 17: analysis of T cell subsets in tumor infiltrating lymphocytes and peripheral T cells
Pharmacodynamic studies revealed that anti-ICOS antibodies STIM001 and STIM003 in the mIgG2a isotype significantly reduced TReg, increased the percentage of CD4+ effector cells and increased the CD4+ effector/TReg ratio and the CD8+/TReg ratio within the Tumor Microenvironment (TME).
The increased CD8+/TReg ratio and increased number of CD4+ effector cells within TME may collectively contribute to the CT26 tumor clearance observed when these anti-ICOS antibodies were co-injected with anti-PDL 1 antibodies in STIM001 and STIM003 efficacy studies (example 11).
Method of producing a composite material
Pharmacodynamic studies were performed in female Balb/c mice carrying CT-26 mouse colon cancer cells (ATCC, CRL-2638). Balb/c mice 6 to 8 weeks old and >18g were supplied by Charles River UK and housed under specific pathogen free conditions. A total of 1X 10E5 CT-26 tumor cells (passage number: P8) were injected subcutaneously in the right flank. All CT-26 tumor bearing animals were assigned to 6 groups (table E17-1) and individual mice were given twice with 200 μ g of antibody or saline (day 13 and day 15 after tumor cell implantation). CD3+ T cells from CT-26 tumor bearing animals were analyzed by FACS on day 16 after tumor cell implantation.
| Group of | Number of animals | Treatment regimens (days 13 and 15 after tumor cell implantation) |
| 1 | 10 | Physiological saline |
| 2 | 10 | anti-ICOS (STIM001) mIgG1 each 200. mu.g |
| 3 | 10 | anti-ICOS (STIM001) mIgG2a each at 200. mu.g |
| 4 | 10 | anti-ICOS (STIM003) mIgG1 each 200. mu.g |
| 5 | 10 | anti-ICOS (STIM003) mIgG2a each at 200. mu.g |
| 6 | 10 | anti-CTLA-4 (9H10) each 200 μ g |
Table E17-1: treatment group
Results
Animals treated with STIM001 and STIM003 of the mIgG2a isotype displayed a lower percentage of CD4+ CD3+ CD45+ cells at the tumor site when compared to the saline treated group (fig. 15A), while STIM001 or STIM003 treatment had a marginal effect on the percentage of CD8+ CD3+ CD45+ cells at the tumor site (fig. 15B). The reduction in CD4+ T cells was attributable to a profound reduction in the effect of the percentage of T regulatory cells in all groups treated with STIM001 and STIM003 antibodies. Notably, animals treated with STIM001 and STIM003 in the mIgG2a isotype exhibited a significant reduction in T-Reg (CD4+ Foxp3+ CD25+) within the TME, whereas STIM001 and STIM003 in the mIgG1 isotype had only a modest effect on T-Reg levels in the TME. In addition, animals treated with STIM001 and STIM003 of the mIgG2a isotype had reduced T-Reg in the TME when compared to animals treated with commercial anti-CTLA-4 (9H10, bibliographic No. 106208, associative Biotech, Inc.) antibodies known to reduce T-Reg [42], but this result did not reach statistical significance (FIG. 15C). The effect of STIM001 and STIM003 in mIgG1 or mIgG2a isotypes on the T-Reg compartment is more specific in the case of Tumor Infiltrating Lymphocytes (TILs). No reduction in T-reg was observed in the periphery (as previously described for anti-CTLA 4[43 ]) (FIG. 15D). Changes in T-Reg levels also resulted in a significant increase in the percentage of intratumoral CD4 effector cells (CD4+ Foxp 3-CD 25-) (fig. 15E), and similarly, the ratio of CD4 effector/T-Reg and the ratio of CD8/T-Reg also increased significantly within the TME in animals treated with STIM001 and STIM003 in mIgG2a (fig. 15F and 15G).
Example 18: effect of anti-ICOS antibodies on the content of ICOS-expressing T cells in CT26 tumor and spleen
Following treatment with anti-ICOS antibodies STIM001 or STIM003, analysis was performed to quantify the percentage of immune cells within the tumor compared to the spleen by analyzing total immune cells in tumor and spleen tissues. STIM001 and STIM003mIgG2a each caused a significant reduction of tregs within the tumor, but not in the spleen, indicating a tumor-selective effect. This depletion is selective for tregs compared to other T cell subtypes. The results presented here help to understand the impact of STIM antibodies on immunity, and confirm that anti-ICOS antibodies with effector function-enabling Fc regions can strongly deplete TReg.
Materials and methods
Mice bearing CT26 tumor were dosed twice with STIM001, STIM003, or anti-CTLA 4 antibody (9H 10). Since anti-CTLA 4antibodies have previously been shown to selectively reduce tregs in tumors, anti-CTLA 4antibodies were included as a positive control for Treg depletion [43 ].
Tumor and immune tissues within the spleen of treated animals were analyzed by FACS after tissue disaggregation.
Details of the FACS antibodies used in this study are shown in table E18. All FACS antibodies were used at the concentrations suggested by the supplier. FACS data was acquired using an Attune NxT flow cytometer and analyzed using FlowJo software.
| Marker substance | Suppliers of goods | Directory number | Batch number | Fluorophores |
| Survival/death | Life technologies Co Ltd | L-34959 | 1784156 | Fixable yellow |
| CD45 | Electronic bioscience | 45-0451-82 | E08336-1636 | PerCp-Cy5.5 |
| CD3 | Electronic bioscience | 48-0032-82 | 4278794 | eFlour 450 |
| CD4 | Electronic bioscience | 11-0042-86 | E0084-1633 | FITC |
| CD8 | Electronic bioscience | 12-0081-85 | E01039-1635 | PE |
| Foxp3 | Electronic bioscience | 17-5773-82 | 4291991 | APC |
| CD25 | Electronic bioscience | 47-0251-82 | 4277960 | APC eF 780 |
| ICOS | Electronic bioscience | 25-9942-82 | E17665-103 | PE-CY7 |
| Fc/Block | Electronic bioscience | 16-0161-86 | E06357-1633 | ------------------ |
Table e18.facs antibodies.
Results
ICOS expression was determined in CT26 tumors and in the spleen of tumor-bearing animals. We observed a percentage increase in tumor infiltrating immune cells expressing ICOS protein (fig. 20), indicating that immune cells in the tumor were more frequently positive for ICOS expression than surrounding immune cells. TReg in the tumors of untreated animals was almost totally (> 90%) positive for ICOS expression, whereas CD8+ effector T cells in the tumors were not (approximately 60%). Comparing the T cell subpopulation in the tumor (also in untreated mice) with the T cell subpopulation in the spleen, a significantly higher (p <0.0001) percentage of intratumoral tregs were positive for ICOS compared to tregs in the spleen and a significantly higher (p <0.001) percentage of intratumoral CD4+ Teff cells were positive for ICOS compared to CD4+ Teff cells in the spleen.
Furthermore, ICOS expression levels on immune cells in the CT26 tumor microenvironment were much higher when compared to immune cells in the spleen in pre-treated mice (fig. 21). ICOS expression was increased on the surface of all subsets of immune cells analyzed in the tumor microenvironment (CD 8T-effector, CD 4T-effector and CD4/FoxP3 Treg cells). It should be noted that although immune cells in both tumor and spleen expressed ICOS, immune cells in tumor (indicated by higher MFI, fig. 21) expressed significantly more ICOS than cells in spleen (indicated by lower MFI, fig. 21). Importantly, TReg in tumors expressed the highest level of ICOS, as previously reported [11 ].
Animals bearing CT26 tumors were treated with 2 doses of antibody STIM001 or STIM003 and with anti-CTLA-4 antibody. STIM antibodies do not affect the overall percentage of CD 45-positive cells (markers of immune cells) in tumors when used in mIgG1 or mIgG2 format. Treatment with these antibodies also did not significantly affect the percentage of CD8 effector T cells in CT26 tumors (figure 22). Treatment with STIM001 in the mIgG2a isotype format resulted in significant (p <0.05) depletion of CD4+ effector T cells, but none of STIM001 mIgG1, STIM003mIgG 1and STIM003mIgG2a affected the percentage of CD4+ effector T cells.
anti-CTLA-4 treatment significantly (although not statistically significantly) increased CD45+ cells and CD8+ effector T cells in TME, but did not affect CD4+ effector T cells (fig. 22).
STIM antibodies significantly affected regulatory T cells in tumors. As shown in figure 23, STIM001 mIgG2a and STIM003mIgG2a significantly and selectively deplete TReg (ICOS expression high) in the tumor microenvironment. Interestingly, the anti-CTLA 4 antibody, although included as a positive control for TReg depletion in this experiment, was also less effective at depleting TReg than STIM mIgG2a antibody.
This selective depletion of TReg results in an increased ratio of CD8 effector T cells to TReg in the tumor and an increased ratio of CD4 effector T cells to TReg in the tumor, both of which should be favorable for an anti-tumor immune response. The ratio data is shown in figure 24.
Such effects were not observed on tregs in the spleen compared to depletion of intratumoral tregs by STIM001 mIgG2a and STIM003mIgG2a (fig. 25, 26, 27). This indicates that the effect of anti-ICOS antibodies on depletion of depleted Treg is not systemic in all tissues. Such selectivity can be advantageous for therapeutic anti-ICOS antibodies when treating a patient's tumor, as preferential depletion of tregs in the tumor microenvironment can selectively mitigate inhibition of anti-tumor effector T cells while minimizing side effects elsewhere in the body. anti-ICOS antibodies may thus promote anti-tumor responses in the immune system, where a low risk of undesirable activation of broader T cell responses may lead to treatment-limiting autoimmune adverse events.
Example 19: antibody stability
The stability of STIM003 human IgG1 was tested during storage, freeze/thaw and purification and was found to retain its stability under all tested conditions. % aggregation was determined by HPLC.
After 3 months of storage in buffer (10mM, 40mM sodium chloride, pH 7.0) at 4 ℃, there was no significant change (> 99%) in the percentage of monomer.
In the heat denaturation test, all samples (n ═ 15) had the same Tm (no significant difference between aliquots) and had comparable heat denaturation curves.
After 3 cycles of freezing and thawing, there was no significant change in Tm (. apprxeq.70.3 ℃), percent monomer, or distribution on SDS-PAGE.
After 7 days of storage at room temperature, there was no significant change in Tm (. apprxeq.70.3 ℃), percent monomer, or distribution on SDS-PAGE.
Protein a recovered 90% after purification.
Example 20: monotherapy efficacy of anti-ICOS Ab against a20 tumor growth in mice
The anti-ICOS antibodies STIM001 mIgG2a and STIM003mIgG2a each showed strong anti-tumor efficacy when used as monotherapy in vivo in the mouse a20 isogenic model.
Materials and methods
Efficacy studies were performed in BALB/c mice using the subcutaneous A20 reticulocytic sarcoma model (ATCC, TIB-208). The A20 cell line is a BALB/c B cell lymphoma line derived from spontaneous reticulocytes found in older BALB/cAnN mice. This cell line has been reported to be positive for ICOSL.
BALB/c mice >18 ng were supplied by Charles River UK and housed without specific pathogens. A total of 1 × 10e 5a 20 cells (passage number below P20) were injected subcutaneously into the right flank of the mice. A20 cells were passaged ex vivo, washed twice in PBS and resuspended in RPMI supplemented with 10% fetal bovine serum. Cell viability was confirmed to be greater than 85% at tumor cell injection. Unless otherwise indicated, antibody or isotype dosing was started from day 8 post tumor cell injection.
STIM001 and STIM003 anti-ICOS antibodies were produced as the mouse IgG2a isotype. Mouse cross-reactive anti-PD-L1 antibody (AbW) was also produced in the same isotype format (mouse IgG2 a). 200 μ g of each of STIM001, STIM003, and anti-PD-L1 antibodies was administered Intraperitoneally (IP) twice a week starting on day 8 after tumor cell implantation (3 weeks between day 8 and day 29). Animal weight and tumor volume were measured 3 times a week starting on the day of tumor cell injection. Tumor volume was calculated by using the modified ellipsoid formula 1/2 (length x width 2). The mice were studied continuously until the mean diameter of the mouse tumor reached 12 mm. The experiment was stopped on day 43 after tumor cell implantation. Tumor growth was monitored and compared to animal tumors treated with isotype control (mIgG2a) antibody. Treatment groups are shown in table E20 below.
| Group of | Number of animals | Treatment regimen (twice weekly for 3 weeks 7 doses) |
| 1 | 8 | mIgG2a isotype control 200. mu.g/mouse/dose |
| 2 | 8 | anti-PD-L1 mIGg2a (AbW)200 ug/mouse/dose |
| 3 | 8 | anti-ICOS mIgG2a STIM 001200. mu.g/mouse/dose |
| 4 | 8 | anti-ICOS mIgG2a STIM 003200. mu.g/mouse/dose |
Table e20. treatment group of the a20 study.
Results
Monotherapy administration of STIM001 or STIM003(mIgG2a) produced a complete anti-tumor response in the a20 tumor model (fig. 32, 33). All animals given STIM001 or STIM003 were cured of the disease. This is in contrast to the results of the isotype control and PD-L1mIgG2a groups (fig. 30, fig. 31). In rare cases, although tumor regression was observed in some animals in the isotype control group (spontaneous regression) and the anti-PDL-1 group, treatment with anti-ICOS antibody produced significantly greater efficacy. At the end of the study, 3/8 control animals and 2/8 anti-PDL-1 treated animals were tumor free. However, all animals treated with STIM001 or STIM003 contained no tumor at the end of the study (8/8 mice in both groups), and showed 100% cure with anti-ICOS antibody.
Example 21: potent Activity of anti-ICOS antibody in combination with anti-PD-L1 antibody in J558 myeloma syngeneic modelIn vivo antitumor efficacy
The anti-ICOS antibody STIM003mIgG2a and the anti-PD-L1 antibody AbW mIgG2a were administered separately and in combination in the J558 tumor model. This is a syngeneic mouse model of myeloma. anti-ICOS antibodies were found to inhibit tumor growth when administered as monotherapy or in combination with anti-PD-L1.
Materials and methods
An anti-tumor efficacy study was performed in Balb/c mice using the subcutaneous J558 plasmacytoma myeloma cell line (ATCC, TIB-6). Supplied by Charles River UK for 6 to 8 weeks of age and>18g of Balb/c mice were housed in a pathogen-free environment. Will be 1X 10 in total6Individual cells (passage number below P20) were injected subcutaneously (100 μ l) into the right flank of the mice. On day 11 post tumor cell injection, animals were randomized and treatment was started based on tumor size, unless otherwise indicated. J558 cells by use of TrypLETMThe expressed enzyme (semer feishell) was passaged ex vivo, washed twice in PBS and resuspended in DMEM supplemented with 10% fetal bovine serum. Cell viability was confirmed to be above 90% at tumor cell injection.
Reaches about 140mm in the tumor3Start treatment at the mean volume of (c). Animals with similar mean tumor sizes were then assigned to 4 groups (see table E-21 for dosing groups). Two antibodies with mouse cross-reactivity were administered twice a week for 3 weeks from day 11 (after tumor cell implantation) (fig. 38), unless the animals had to be removed from the study due to welfare (rarity) or tumor size. As a control, a group of animals (n ═ 10) was administered while using a physiological saline solution. For the combination group, both STIM003 and anti-PDL 1 antibodies were administered at 60 μ g and 200 μ g: (A), (B), (C), (D), (In 0.9% normal saline) IP in parallel. Tumor growth was monitored over 37 days and compared to tumors in animals treated with saline. Animal weight and tumor volume were measured 3 times a week starting on the day of tumor cell injection. By using the modified ellipsoid formula 1/2 (length x width)2) The tumor volume was calculated. The mice were studied continuously until the mice reached a tumor of 12mm3Or in rare cases until the onset of tumor ulceration (welfare) is observed.
Table e21. treatment group for J558 efficacy study
Results
J558 syngeneic tumors were highly aggressive and all animals in the saline control group (n ═ 10) had to be removed from the study by day 21 due to tumor size. Both anti-STIM 003mIgG2a and anti-PDL 1mIgG2a showed good efficacy as monotherapies in this model, with 37.5% and 75% of the animals cured the disease, respectively. Importantly, the combination of both antibodies allowed 100% of the animals to shed plasmacytoma tumors by day 37. The data is shown in fig. 38.
Example 22: administration of anti-PD 1 significantly increased ICOS expression on TIL more than anti-PD-L1 antibody
Pharmacodynamic studies were performed in animals bearing established CT26 tumors to assess the effect of treatment with anti-PD-L1 or anti-PD-1 antibodies on ICOS expression on Tumor Infiltrating Lymphocyte (TIL) subpopulations. The following antibodies were compared:
anti-PD-L1 AbW mIgG1[ Limited Effector Functions ]
anti-PD-L1 AbW mIgG2a [ having effector function ]
anti-PD-L110F9. G2 rat IgG2b [ with Effector function ]
anti-PD 1 antibody RMT1-14 rat IgG2a [ Effector null ].
Tumors from treated mice were isolated, dissociated into single cells and stained for CD45, CD3, CD4, CD8, FOXP3 and ICOS.
Materials and methods
Rat anti-PD-1 RMP1-14 IgG2a (BioXCell; catalog No.: BE0146), rat anti-PD-L110F9. G2IgG2b (Lenovo Biotech Co., Ltd.; catalog No.: 124325) and anti-PD-L1 AbW mIgG 1and mIgG2a were tested in the CT26 tumor model by intraperitoneal administration at 130. mu.g on days 13 and 15 after tumor cell implantation. On day 16, animals were picked and mouse tumors harvested for FACS analysis. Tumors were dissociated and homogenized using a mouse tumor dissociation kit (Miltenyi Biotec). The resulting cell suspension was clarified through a 70 μ M filter, pelleted and resuspended at 2 million cells/well in FACS buffer in a 96-well culture plate. The cell suspension was incubated with anti-16/32 mAb (electronic biosciences) and stained with FACS antibodies specific for all of CD3(17A2), CD45(30-F11), CD4(RM4-5), CD8(53-6.7) and ICOS (7E.17G9) obtained from electronic biosciences, Inc. Cells were also stained with LiveDead yellow fixable viability dye (life technologies). For intracellular staining of Foxp3, samples were fixed, permeabilized and stained with an antibody specific for Foxp3 (electron biosciences, FJK-16 s). The samples were resuspended in PBS and the data were taken on an Attune flow cytometer (invitrogen) and analyzed using FlowJo V10 software (Treestar).
Results
Treatment with anti-PD 1and anti-PD-L1 antibodies resulted in only marginal increases in the percentage of ICOS-expressing CD8 cells and T regs at the time points measured. However, a clear and significant (over saline treated group) increase in ICOS expression (increased dMFI) was observed on the ICOS + ve CD8 cell surface in response to anti-PD 1 rat IgG2 a. Upregulation of ICOS expression on CD4 effector and CD 4T Reg cells was also noted, although this was not statistically significant. This anti-PD 1 antibody induced a significant increase in ICOS expression on CD8 effector cells, which was rare in the case of anti-PD-L1 mIgG2 a. Similarly, when comparing different forms of anti-PD-L1 antibodies, it is rare to observe that in some treated animals the antibody with the lowest effector function (mIgG1) correlates with higher ICOS expression on effector CD8 and CD4 cells when compared to antibodies with effector function (mIgG2a and ratIgG2 b). See fig. 39.
Increased ICOS expression on effector CD8/CD 4T cells may have the effect of making these cells more sensitive to depletion by anti-ICOS antibodies (e.g., to treatment of mice with STIM003mIgG2 a). Antibodies exhibiting lower ICOS induction in effector CD8 and CD 4T cells may be preferred for use in combination with anti-ICOS antibodies. Data from this study indicate that anti-PD-L1 effector-positive antibodies may be particularly useful in combination with anti-ICOS effector-positive antibodies, reflecting the anti-tumor efficacy observed when combining anti-PDL 1mIgG2a, reported in other examples herein, with STIM003mIgG2 a.
Example 23: potentiation of in vivo single dose anti-ICOS antibody monotherapy in a B cell lymphoma syngeneic modelAnti-tumor efficacy
This experiment confirmed the anti-tumor efficacy of STIM003mIgG2a as a monotherapy. Exhibits potent anti-tumor efficacy after brief exposure to STIM003mIgG2 a.
Materials and methods
Efficacy studies were performed in BALB/c mice using the subcutaneous A20 reticulocytic sarcoma model (ATCC accession number CRL-TIB-208). Supplied by Charles River UK for 6 to 8 weeks of age and>18g of Balb/c mice were housed in a pathogen-free environment. A total of 1 × 10E 5a 20 cells (passage number below P20) were injected subcutaneously into the right flank of the mice. Treatment was started on day 8 after tumor cell injection as shown in the table below. A20 cells were obtained by using trypLETMThe expressed enzyme (semer feishell) was passaged ex vivo, washed twice in PBS, and resuspended in RPMI supplemented with 10% fetal bovine serum. Injection in tumor cellsCell viability was confirmed to be above 85%. STIM003mIgG2a was used in a Single Dose (SD) of 60 μ g (equivalent to 3mg/kg for 20g animals) or in multiple doses of 60 μ g (MD, twice a week for 3 weeks). The anti-tumor efficacy observed in response to both protocols was compared to that of animals "treated" with saline (MD, twice a week for 3 weeks). The antibody was administered Intraperitoneally (IP) at 1mg/ml in 0.9% physiological saline. Animal weight and tumor volume were coughed 3 times a week from the day of tumor cell injection. By using the modified ellipsoid formula 1/2 (length x width)2) The tumor volume was calculated. The mice were studied continuously until the mice reached a tumor of 12mm3Or rarely until tumor ulceration is observed (welfare).
| Group of | Number of animals | Treatment protocol (IP injection) |
| 1 | 10 | Physiological saline (multiple doses starting on day 8, twice a week for 3 weeks) |
| 2 | 10 | STIM003mIgG2A (multiple doses starting on day 8, twice a week for 3 weeks) |
| 3 | 10 | STIM003mIgG2A (single dose on day 8) |
TABLE E23-1 treatment groups.
Results
Both multiple and single doses of STIM003mIgG2a produced potent and significant monotherapy anti-tumor efficacy as shown by the number of animals with no evidence of tumor growth at the endpoint (day 41). SD cured 7/10 animals for disease, while multiple doses injected with a20B cell lymphoblasts cured 9/10 animals. All animals in the saline treated group had to be removed from the study by day 40 due to tumor size. See fig. 40.
Humane endpoint survival statistics were calculated from the kaplan-meier curve (figure 41) using GraphPad Prism V7.0. This method was used to determine whether treatment was associated with improved survival. The risk ratio (Mantel-Haenszel) values and their associated P-values (log rank Mantel-Cox) are shown in the table below.
Table E23-1. risk ratio (Mantel-hurs) values and their associated P values (log rank Mantel-Cox) corresponding to the kaplan-meier curve of fig. 41.
Example 24: time and dose dependent effects of anti-ICOS antibodies in CT-26 tumor bearing animals
This example presents the results of a pharmacodynamic study to assess the effect of anti-ICOS antibodies on immune cells in CT-26 tumor-bearing mice. T cells and B cell subsets from different tissues were analyzed by FACS after a single dose of STIM003mIgG2 a.
Method of producing a composite material
Animals bearing CT-26 tumors were dosed intraperitoneally with saline or 200 μ g, 60 μ g, or6 μ g STIM003 after tumor cell implantation at day 12 after treatment tumor tissue, blood, Tumor Draining Lymph Nodes (TDLN), and spleen were collected at day 1, day 2, day 3, day 4, and day 8 post-treatment tumor dissociation kit (american and biotechnology) to form single cell suspensions, spleen tissue was dissociated using mild MACS dissociation, red blood cells were lysed using RBC lysis buffer, tumor draining lymph nodes were mechanically disaggregated to form single cell suspensions, the resulting cell suspensions were clarified by tissue through 70 μ M or 40 μ M filters, then cells were washed twice in RMPI complete medium and finally resuspended in ice-cold FACS buffer, whole blood was collected into plasma tubes and red blood was lysed using lysis buffer, cells were washed twice in RMPI complete medium and finally resuspended in ice-cold FACS buffer, cells were resuspended in plasma tubes and lysed with lysis buffer for single cell staining with CD 19-CD 96, CD8, CD 96, CD 8-CD 96, CD 8-CD 96, CD8, CD 96, CD8, CD.
The results are presented and discussed below.
Higher ICOS expression on intratumoral T-reg in CT26 model
When comparing the percentage of ICOS expressing Tumor Infiltrating Lymphocytes (TILs) with the percentage of immune cells in spleen, blood and TDLN, we demonstrated thatMore immune cells in the CT-26 tumor microenvironment expressed ICOS relative to other tissues. More importantly, the percentage of ICOS positive T-reg cells in all tissues and at all time points was higher than the percentage of CD4 or CD8 effector T cells positive for ICOS. Importantly, the mfi (relative expression) of ICOS also follows a similar ordering of expression, with intratumoral T-reg being highly positive for ICOS expression relative to other TIL subtypes. Interestingly, the ICOS was performed within the timeframe of this experiment+The percentage of TIL did not change significantly. Similar results were seen in spleen and TDLN. On the other hand, ICOS expression on T effector cells was relatively stable but increased on T-reg during the experiment in blood. The data fully demonstrate that more cells express ICOS in the tumor microenvironment and that these positive cells also express more ICOS molecules on their surface. More importantly, T reg in TIL is highly positive for ICOS. See fig. 42.
Greatly depleting intratumoral T-reg cells in response to STIM003 administration
In response to the STIM003mIgG2a antibody, T-reg cell depletion (CD4+ CD25+ Foxp3) in TME was extremely large and rapid. Since T-reg has a high ICOS expression compared to other T cell subsets, it is expected that anti-ICOS antibodies with effector functions will preferentially deplete these cells. T-reg was depleted continuously at lower doses of STIM003 (6 μ g corresponds to 0.3mg/kg in 20g animals) and most of the T-reg was depleted from TME by day 3. Interestingly, by day 8, T-reg cell reconstitution TME subsequently reached levels slightly higher than those observed in saline treated animals. Reconstitution of T-reg cells at lower doses can be attributed to an increase in proliferating CD 4T cells in TME, as evidenced by the observed increase in Ki-67+ CD 4T cells. T-Reg was chronically depleted in TME at doses above 6 μ g, as demonstrated by complete T Reg depletion up to the last time point analyzed in this study (day 8). However, in blood, T-reg cells were transiently depleted at all doses. Importantly, by day 8, all treated animals had similar (or higher for the 6 μ g dose) levels of T-reg cells in the blood as compared to saline treated animals. The data are shown in figure 43. Notably, and similar to the previously published data for depletion of CTLA-4antibodies, there was no significant change in the percentage of T-reg cells in spleen or TDLN tissues, indicating that T-reg cells could be spared from depletion in these organs.
Overall, a significant depletion of T-reg cells in TME was achieved in the CT-26 model at doses as low as 6 micrograms/animal. However, a dose of 60 μ g resulted in long-term depletion of up to 8 days after injection of STIM003mIgG2 a. This cannot be improved by using higher doses (200 μ g).
STIM003mIgG2a increased the CD8: T Reg ratio and the CD4: T Reg ratio
The effect of STIM003 on the T-eff: T-reg ratio is shown in FIG. 44.
STIM003mIgG2a increased the CD8: T-reg ratio and the CD4 eff: T-reg ratio. Although all treatment doses were associated with an increase in the ratio of T-eff to T-reg, a moderate dose of 60 μ g (equivalent to 3mg/kg in 20g animals) was associated with the highest ratio by day 8 post-treatment.
Interestingly, the rate was higher up to day 4 at the 6 μ g dose, but matched that of saline treated animals by day 8 post-treatment. This can be explained by reconstructing the TReg observed for this dose by day 8 post-treatment. On the other hand, at doses of 60 μ g or 200 μ g, the Teff to T-reg ratio remained high at all time points. This is explained by the long-term depletion of tregs at these doses. Notably, at higher doses (200 μ g), the rate improved only moderately by day 8 despite long-term Treg depletion. This can be achieved by depleting some ICOS at high concentrations of STIM003INTEffector cells.
In summary, the data show a decrease in TReg and an increased effector, the T reg ratio, at all doses tested. However, an optimal dose of 60 μ g (about 3mg/kg) achieves both a long-term depletion of T-reg and the highest ratio of T-eff to T-reg, which would correlate with the most favorable immune environment for eliciting an anti-tumor immune response. Interestingly, a similar pattern was observed in blood, where a moderate dose of 60 μ g correlated with the highest ratio of T-eff to T-reg. Importantly, in the blood, an improvement in the ratio was observed at earlier time points (between day 3 and day 4).
Effector cell activation in response to STIM003
CD107a expresses on the surface of tumor infiltrating T effector cells a reliable marker previously identified as cells that have been activated and exert cytotoxic activity [44 ]. In this study, this marker was used to confirm that STIM003, in addition to depleting T-reg, can stimulate cytotoxic activity of effector T cells in TME. Interestingly, on day 8 post-treatment, surface expression of CD107a was increased on both CD4 and CD8 effector T cell compartments at all doses of STIM 003. Furthermore, since no improvement was seen with 200 μ g administration, this up-regulation of CD107a expression on the surface of both CD4 and CD 8T cells appeared to plateau when animals were administered with 60 μ g.
To further demonstrate the activation of effector cells in the TME, cytokine release by CD4 and CD8 TIL was analyzed by FACS as expected and consistent with the in vitro agonistic data presented in earlier examples herein, STIM003mIgG2a at all doses promoted the production of pro-inflammatory cytokines IFN- γ and TNF- α by effector CD4 and CD 8T cells the induction of pro-inflammatory cytokine production appeared to be higher at the optimal dose of 60 μ g indeed, 60 μ g of STIM003 significantly increased cytokine production by CD 4T cells similar trends were seen for pro-inflammatory cytokines IFN- γ and TNF- α produced by effector CD 8T cells in the TME the data are shown in figure 45.
Overall, STIM003 at all doses resulted in T cell activation in the TME as demonstrated by (1) the presentation of the degranulation marker CD107a on its surface and (2) the production of Th1 cytokines (IFN γ and TNF α) by T cells, indicating that STIM003 largely affected the immune environment in the TME and served a dual role in depleting Treg cells and stimulating the killing activity of T effector cells.
Human dose estimation
Based on preclinical efficacy data seen in mice, initial predictions can be made from clinical doses appropriate for human patients, based on the corresponding biological surface regions (BSA) [45 ].
For example, considering that the optimal anti-ICOS IgG dose in mice is 3mg/kg (60 μ g) and following the method of reference [45], the corresponding dose for humans is 0.25 mg/kg.
Using Mosler's equation, BSA was 1.68m for 60kg and 1.70m individuals2. Multiplying the dose in mg/kg by a factor of 35.7(60/1.68) gives a fixed dose of 15 mg. For an individual of 80kg, the corresponding fixed dose would be 20 mg.
The dosage can be optimized for human therapy in clinical trials to determine a safe and effective treatment regimen.
Example 25: bioinformatic analysis of data from tumor samples
One target group of cancers according to the invention are those associated with relatively high levels of ICOS + immunosuppressive tregs.
To identify cancer types associated with high levels of tregs, transcriptome data were obtained from The cancer genomic map (TCGA) public dataset and analyzed for ICOS and FOXP3 expression levels. TCGA is a large-scale study with catalogued genomic and transcriptomics data accumulating many different types of cancer and includes mutations, copy number variation, mRNA and miRNA gene expression and DNA methylation, as well as extensive sample metadata.
Gene Set enrichment analysis (Gene Set enrichment analysis, GSEA) was performed as follows. Gene expression RNA sequence data collected as part of the TCGA consortium was downloaded from the UCSC xeno functional genomics browser as log2(normalized _ count + 1). Non-tumor tissue samples were removed from the dataset, leaving data for 20530 genes from 9732 samples. The algorithm from [46] and its implementation in [47] to calculate enrichment scores for genes within a given gene set was used to transpose gene level counts to the gene set score for each sample. The gene set of interest was defined to contain both ICOS and FOXP 3. Samples were grouped by primary disease and the ssGSEA scores for each group were compared within 33 primary disease groups. The disease groups that were found to show the highest median scores were lymphoid neoplasm diffuse large B-cell lymphoma, thymoma, head and neck squamous cell carcinoma, although diffuse large B-cell lymphoma showed a multimodal distribution with a subset of high scores and a score lower than the rest of the group median scores.
In the ranking of the highest to lowest ssGSEA scores for ICOS and FOXP3 expression, the top 15 cancer types were:
diffuse large B-cell lymphoma of DLBC (n ═ 48) lymphoid neoplasm
THYM (n-120) thymoma
HNSC (n-522) head and neck squamous cell carcinoma
TGCT (n 156) testicular germ cell tumor
STAD (n-415) gastric adenocarcinoma
SKCM (n 473) skin melanoma
CESC (n ═ 305) cervical squamous cell carcinoma and endocervical adenocarcinoma
LUAD (n 517) lung adenocarcinoma
LAML (n 173) acute myeloid leukemia
ESCA (n 185) esophageal cancer
LUSC (n-502) squamous cell carcinoma of lung
READ (n ═ 95) rectal adenocarcinoma
COAD (n-288) Colon adenocarcinoma
BRCA (n-1104) invasive carcinoma of the breast
LIHC (n ═ 373) liver hepatocellular carcinoma
Where n is the number of patient samples for the cancer type in the TCGA dataset. The anti-ICOS antibodies described herein are useful for treating these and other cancers.
Cancers associated with relatively high levels of ICOS + immunosuppressive tregs and further expressing PD-L1 may respond particularly well to treatment with anti-ICOS antibodies in combination with anti-PD-L1 antibodies. For this purpose, appropriate therapeutic regimens and antibodies have been detailed in the foregoing description.
Using the TCGA dataset as described previously, the enrichment scores for ICOS and FOXP3 were related to the expression level of PD-L1 using Spearman's rank correlation (Spearman's correction) and grouped by primary disease indication. P-values were calculated for each group and a P-value of 0.05 (corrected with Bonferroni's multiplex comparison correction) was considered statistically significant. . The disease groups with the highest correlation between ICOS/FOXP3 and PD-L1 expression were:
TGCT (n 156) testicular germ cell tumor
COAD (n-288) Colon adenocarcinoma
READ (n ═ 95) rectal adenocarcinoma
BLCA (n 407) urothelial carcinoma of bladder
OV (n-308) ovarian serous cystadenocarcinoma
BRCA (n-1104) invasive carcinoma of the breast
SKCM (n 473) skin melanoma
CESC (n ═ 305) cervical squamous cell carcinoma and endocervical adenocarcinoma
STAD (n-415) gastric adenocarcinoma
LUAD (n 517) lung adenocarcinoma
Patients may be selected for treatment based on an assay that determines that the patient's cancer is associated with expression of ICOS + immunosuppressive tregs and PD-L1. For cancer types (where there is a high correlation score as described above), it may be sufficient to determine the presence (e.g., above a threshold) of one of ICOS + immunosuppressive tregs and PD-L1 expression. PD-L1 immunohistochemical analysis may be used in this context.
Example 26: assessment of other anti-ICOS antibodies
The CL-74570 and CL-61091 antibody sequences identified in example 12 were synthesized and expressed in HEK cells in the form of IgG 1.
Functional characterization of these antibodies was performed using HTRF analysis similar to that described in example 6, with modifications to adapt the analysis to purified IgG1 rather than to BCT supernatant. BCT supernatant was replaced with 5 μ L of supernatant containing human IgG1 antibody expressed from HEK cells and the total volume was brought to 20 μ L/well using HTRF buffer as described previously. Human IgG1 antibody was used as a negative control. Both antibodies exhibited greater than 5% effect on binding to human and mouse ICOS (as calculated using equation 1) and thus confirmed positive tests in this analysis.
The ability of these antibodies to bind human and mouse ICOS expressed on the surface of CHO-S cells was further confirmed using a microscopic ball assay (Mirrorball assay). In this assay, 5. mu.l of supernatant containing anti-ICOS IgG1 was transferred to each well of 384-mirror-ball black plates (Corning). Binding of anti-ICOS antibodies was detected by adding 10 μ l of goat anti-human 488 (jackson immunoresearch) diluted in assay buffer (PBS + 1% BSA + 0.1% sodium azide) at a concentration of 0.8mg/ml to all wells.
For positive control wells, 5 μ L of reference antibody diluted to 2.2 μ g/mL in assay medium was added to the plate. For negative control wells, 5 μ l of hybridization control IgG1 diluted to 2.2 μ g/mL in assay medium was added to the plates. Add 10. mu.M of DRAQ5 (Saimer Feishell science) to 0.4X 10 resuspended in assay buffer6One/ml of cells and 5 μ l was added to all wells. The plates were incubated at 4 degrees for 2 hours.
Fluorescence intensity was measured using a vitroscope reader (TTP Leiberttaceae (TTP Labtech)) and Alexafluor488 (excitation 493nm, emission 519nm) was measured from 500-. The analytical signal was measured as the mean intensity of the median (FL 2).
The total binding was defined using a reference antibody at an assay concentration of 2.2 μ g/mL. Nonspecific binding was defined using the hybridization control hIgG1 at an assay concentration of 2.22. mu.g/mL. Both antibodies exhibited greater than 1% effect and thus confirmed positive tests in this assay.
Each of CL-74570 and CL-61091 also demonstrated binding to human and mouse ICOS expressed on CHO-S cells as determined by flow cytometry. FACS screening was performed using a method similar to that described in example 6, with modifications to adapt the analysis to purified IgG1 instead of BCT supernatant. Both antibodies exhibited > 10-fold binding over the geometric mean of negative controls bound to hICOS, mcios and WT CHO cells.
Tables E26-1, CL-74570 and CL 61091 for functional characterization.
Reference to the literature
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Sequence of
Antibody STIM001
VH domain nucleotide sequence: 367 SEQ ID NO
CAGGTTCAGGTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTTCCACCTTTGGTATCACCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAATGGATGGGATGGATCAGCGCTTACAATGGTGACACAAACTATGCACAGAATCTCCAGGGCAGAGTCATCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTTTATTACTGTGCGAGGAGCAGTGGCCACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA
VH domain amino acid sequence: 366 SEQ ID NO
QVQVVQSGAEVKKPGASVKVSCKASGYTFSTFGITWVRQAPGQGLEWMGWISAYNGDTNYAQNLQGRVIMTTDTSTSTAYMELRSLRSDDTAVYYCARSSGHYYYYGMDVWGQGTTVTVSS
VH CDR1 amino acid sequence: GYTFSTFG SEQ ID NO:363
VH CDR2 amino acid sequence: ISAYNGDT SEQ ID NO:364
VH CDR3 amino acid sequence: ARSSGHYYYYGMDV SEQ ID NO:365
VL domain nucleotide sequence: 374 SEQ ID NO
GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGAATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTTTTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCACCAGAGTGGAGGCTGAGGATGTTGGAATTTATTACTGCATGCAATCTCTACAAACTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA
VL domain amino acid sequence: 373 SEQ ID NO
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNEYNYLDWYLQKPGQSPQLLIFLGSNRASGVPDRFSGSGSGTDFTLKITRVEAEDVGIYYCMQSLQTPLTFGGGTKVEIK
VL CDR1 amino acid sequence: QSLLHSNEYNY SEQ ID NO:370
VL CDR2 amino acid sequence: LGS SEQ ID NO 371
VL CDR3 amino acid sequence: MQSLQTPLT SEQ ID NO:372
Antibody STIM002
VH domain nucleotide sequence: 381 SEQ ID NO
CAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTAGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGATCTACGTATTTCTATGGTTCGGGGACCCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA
VH domain amino acid sequence: 380 SEQ ID NO
QVQLVQSGGEVKKPGASVKVSCKASGYTFTSYGFSWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSTYFYGSGTLYGMDVWGQGTTVTVSS
VH CDR1 amino acid sequence: GYTFTSYG SEQ ID NO:377
VH CDR2 amino acid sequence: ISAYNGNT SEQ ID NO:378
VH CDR3 amino acid sequence: ARSTYFYGSGTLYGMDV SEQ ID NO:379
VL domain nucleotide sequence: 388
GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTGATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCGGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAA
Corrected STIM002 VL domain nucleotide sequence: 519 SEQ ID NO
GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTGATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCGGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGCTCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAA
VL domain amino acid sequence: 387 SEQ ID NO
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGYNYLDWYLQKPGQSPQLLIYLGSTRASGFPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPLSFGQGTKLEIK
VL CDR1 amino acid sequence: QSLLHSDGYNY SEQ ID NO:384
VL CDR2 amino acid sequence: LGS SEQ ID NO 385
VL CDR3 amino acid sequence: MQALQTPLS SEQ ID NO:386
Antibody STIM002-B
VH domain nucleotide sequence: 395 of SEQ ID NO
CAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTAGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGATCTACGTATTTCTATGGTTCGGGGACCCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA
VH domain amino acid sequence: 394 SEQ ID NO
QVQLVQSGGEVKKPGASVKVSCKASGYTFTSYGFSWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSTYFYGSGTLYGMDVWGQGTTVTVSS
VH CDR1 amino acid sequence: GYTFTSYG SEQ ID NO:391
VH CDR2 amino acid sequence: ISAYNGNT SEQ ID NO:392
VH CDR3 amino acid sequence: ARSTYFYGSGTLYGMDV SEQ ID NO:393
VL domain nucleotide sequence: 402 SEQ ID NO
GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTGATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCGGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAA
VL domain amino acid sequence: 401 of SEQ ID NO
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGYNCLDWYLQKPGQSPQLLIYLGSTRASGFPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPCSFGQGTKLEIK
VL CDR1 amino acid sequence: QSLLHSDGYNC SEQ ID NO:398
VL CDR2 amino acid sequence: LGS SEQ ID NO 399
VL CDR3 amino acid sequence: MQALQTPCS SEQ ID NO:400
Antibody STIM003
VH domain nucleotide sequence: 409 SEQ ID NO
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGAGTCACCTTTGATGATTATGGCATGAGCTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGARTGGGTCTCTGGTATTAATTGGAATGGTGGCGACACAGATTATTCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTACAAATGAATAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGAGGGATTTCTATGGTTCGGGGAGTTATTATCACGTTCCTTTTGACTACTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCA
Corrected STIM003 VH domain nucleotide sequence: 521 of SEQ ID NO
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGAGTCACCTTTGATGATTATGGCATGAGCTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGAGTGGGTCTCTGGTATTAATTGGAATGGTGGCGACACAGATTATTCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTACAAATGAATAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGAGGGATTTCTATGGTTCGGGGAGTTATTATCACGTTCCTTTTGACTACTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCA
VH domain amino acid sequence: 408 SEQ ID NO
EVQLVESGGGVVRPGGSLRLSCVASGVTFDDYGMSWVRQAPGKGLEWVSGINWNGGDTDYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARDFYGSGSYYHVPFDYWGQGILVTVSS
VH CDR1 amino acid sequence: GVTFDDYG SEQ ID NO:405
VH CDR2 amino acid sequence: INWNGGDT SEQ ID NO:406
VH CDR3 amino acid sequence: ARDFYGSGSYYHVPFDY SEQ ID NO:407
VL domain nucleotide sequence: 416 SEQ ID NO
GAAATTGTGTTGACGCAGTCTCCAGGGACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGAAGCTACTTAGCCTGGTACCAGCAGAAACGTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCGATGGGTCTGGGACAGACTTCACTCTCTCCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCACCAGTATGATATGTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA
VL domain amino acid sequence: 415 SEQ ID NO
EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKRGQAPRLLIYGASSRATGIPDRFSGDGSGTDFTLSISRLEPEDFAVYYCHQYDMSPFTFGPGTKVDIK
VL CDR1 amino acid sequence: QSVSRSY SEQ ID NO:412
VL CDR2 amino acid sequence: GAS SEQ ID NO 413
VL CDR3 amino acid sequence: HQYDMSPFT SEQ ID NO:414
Antibody STIM004
VH domain nucleotide sequence:
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGACTCACCTTTGATGATTATGGCATGAGCTGGGTCCGCCAAGTTCCAGGGAAGGGGCTGGAGTGGGTCTCTGGTATTAATTGGAATGGTGATAACACAGATTATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGAGGGATTACTATGGTTCGGGGAGTTATTATAACGTTCCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCASEQ ID NO:423
VH domain amino acid sequence:
EVQLVESGGGVVRPGGSLRLSCAASGLTFDDYGMSWVRQVPGKGLEWVSGINWNGDNTDYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARDYYGSGSYYNVPFDYWGQGTLVTVSS SEQ ID NO:422
VH CDR1 amino acid sequence: GLTFDDYG SEQ ID NO:419
VH CDR2 amino acid sequence: INWNGDNT SEQ ID NO:420
VH CDR3 amino acid sequence: ARDYYGSGSYYNVPFDY SEQ ID NO:421
VL domain nucleotide sequence: 431 SEQ ID NO
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATATATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGAAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGTTCACCATTCACTTCGGCCCTGGGACCAAAGTGGATATCAAA
Such as a VL domain amino acid sequence encoded by the above VL domain nucleotide sequence.
Corrected VL domain nucleotide sequence: 430 SEQ ID NO
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATATATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGAAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGTTCACCATTCTTCGGCCCTGGGACCAAAGTGGATATCAAA
Corrected VL domain amino acid sequence: 432 SEQ ID NO
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIRRLEPEDFAVYYCQQYGSSPFFGPGTKVDIK
VL CDR1 amino acid sequence: QSVSSSY SEQ ID NO:426
VL CDR2 amino acid sequence: 427 GAS SEQ ID NO
VL CDR3 amino acid sequence: QQYGSSPF SEQ ID NO:428
Antibody STIM005
VH domain nucleotide sequence: 439 of SEQ ID NO
CAGGTTCAGTTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTAATAGTTATGGTATCATCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGTTCACAATGGTAACACAAACTGTGCACAGAAGCTCCAGGGTAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGAACTGACGACACGGCCGTGTATTACTGTGCGAGAGCGGGTTACGATATTTTGACTGATTTTTCCGATGCTTTTGATATCTGGGGCCACGGGACAATGGTCACCGTCTCTTCA
VH domain amino acid sequence: 438 SEQ ID NO
QVQLVQSGAEVKKPGASVKVSCKASGYTFNSYGIIWVRQAPGQGLEWMGWISVHNGNTNCAQKLQGRVTMTTDTSTSTAYMELRSLRTDDTAVYYCARAGYDILTDFSDAFDIWGHGTMVTVSS
VH CDR1 amino acid sequence: GYTFNSYG SEQ ID NO:435
VH CDR2 amino acid sequence: ISVHNGNT SEQ ID NO:436
VH CDR3 amino acid sequence: ARAGYDILTDFSDAFDI SEQID NO:437
VL domain nucleotide sequence: 446 SEQ ID NO
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAACATTAATAACTTTTTAAATTGGTATCAGCAGAAAGAAGGGAAAGGCCCTAAGCTCCTGATCTATGCAGCATCCAGTTTGCAAAGAGGGATACCATCAACGTTCAGTGGCAGTGGATCTGGGACAGACTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACATCTGTCAACAGAGCTACGGTATCCCGTGGGTCGGCCAAGGGACCAAGGTGGAAATCAAA
VL domain amino acid sequence: 445 SEQ ID NO
DIQMTQSPSSLSASVGDRVTITCRASQNINNFLNWYQQKEGKGPKLLIYAASSLQRGIPSTFSGSGSGTDFTLTISSLQPEDFATYICQQSYGIPWVGQGTKVEIK
VL CDR1 amino acid sequence: QNINNF SEQ ID NO:442
VL CDR2 amino acid sequence: AAS SEQ ID NO 443
VL CDR3 amino acid sequence: QQSYGIPW SEQ ID NO:444
Antibody STIM006
VH domain nucleotide sequence: 453 or more
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTTCATGAGCTGGATCCGCCAGGCGCCAGGGAAGGGGCTGGAGTGGATTTCATACATTAGTTCTAGTGGTAGTACCATATACTACGCAGACTCTGTGAGGGGCCGATTCACCATCTCCAGGGACAACGCCAAGTACTCACTGTATCTGCAAATGAACAGCCTGAGATCCGAGGACACGGCCGTGTATTACTGTGCGAGAGATCACTACGATGGTTCGGGGATTTATCCCCTCTACTACTATTACGGTTTGGACGTCTGGGGCCAGGGGACCACGGTCACCGTCTCCTCA
VH domain amino acid sequence: 454 SEQ ID NO
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYFMSWIRQAPGKGLEWISYISSSGSTIYYADSVRGRFTISRDNAKYSLYLQMNSLRSEDTAVYYCARDHYDGSGIYPLYYYYGLDVWGQGTTVTVSS
VH CDR1 amino acid sequence: GFTFSDYFSEQ ID NO:449
VH CDR2 amino acid sequence: ISSSGSTI SEQ ID NO:450
VH CDR3 amino acid sequence: ARDHYDGSGIYPLYYYYGLDV SEQ ID NO:451
VL domain nucleotide sequence: 460 SEQ ID NO
ATTGTGATGACTCAGTCTCCACTCTCCCTACCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTATTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTTATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGCAGTTTTGGCCAGGGGACCACGCTGGAGATCAAA
VL domain amino acid sequence: 459 SEQ ID NO
IVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDYYLQKPGQSPQLLIYLGSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRSFGQGTTLEIK
VL CDR1 amino acid sequence: QSLLHSNGYNY SEQ ID NO:456
VL CDR2 amino acid sequence: lgs SEQ ID NO 457
VL CDR3 amino acid sequence: MQALQTPRS SEQ ID NO:458
Antibody STIM007
VH domain nucleotide sequence: 467 SEQ ID NO
CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAGACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACTACTGGAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTGGAGTGGCTTGCAGTCATTTATTGGGATGATGATAAGCGCTACAGCCCATCTCTGAAGAGCAGACTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTTCTGTACACACGGATATGGTTCGGCGAGTTATTACCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA
VH domain amino acid sequence: 466 of SEQ ID NO
QITLKESGPTLVKPTQTLTLTCTFSGFSLSTTGVGVGWIRQPPGKALEWLAVIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYFCTHGYGSASYYHYGMDVWGQGTTVTVSS
VH CDR1 amino acid sequence: GFSLSTTGVG SEQ ID NO:463
VH CDR2 amino acid sequence: IYWDDDK SEQ ID NO:464
VH CDR3 amino acid sequence: THGYGSASYYHYGMDV SEQ ID NO:465
VL domain nucleotide sequence: 474 SEQ ID NO
GAAATTGTATTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACCAACTACTTAGCCTGGCACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCACCGTAGCAACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC
VL domain amino acid sequence: 473 SEQ ID NO
EIVLTQSPATLSLSPGERATLSCRASQSVTNYLAWHQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHRSNWPLTFGGGTKVEIK
VL CDR1 amino acid sequence: QSVTNY SEQ ID NO:470
VL CDR2 amino acid sequence: DAS SEQ ID NO:471
VL CDR3 amino acid sequence: QHRSNWPLT SEQ ID NO:472
Antibody STIM008
VH domain nucleotide sequence: 481 SEQ ID NO
CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAGACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACTAGTGGAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTGGAGTGGCTTGCAGTCATTTATTGGGATGATGATAAGCGCTACAGCCCATCTCTGAAGAGCAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTTCTGTACACACGGATATGGTTCGGCGAGTTATTACCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA
VH domain amino acid sequence: 480 SEQ ID NO
QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLAVIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYFCTHGYGSASYYHYGMDVWGQGTTVTVSS
VH CDR1 amino acid sequence: GFSLSTSGVG SEQ ID NO:477
VH CDR2 amino acid sequence: IYWDDDK SEQ ID NO:478
VH CDR3 amino acid sequence: THGYGSASYYHYGMDV SEQ ID NO:479
VL domain nucleotide sequence: 488 of SEQ ID NO
GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACCAACTACTTAGCCTGGCACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA
VL domain amino acid sequence: 489 SEQ ID NO
EIVLTQSPATLSLSPGERATLSCRASQSVTNYLAWHQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK
VL CDR1 amino acid sequence: QSVTNY SEQ ID NO:484
VL CDR2 amino acid sequence: DAS SEQ ID NO of 485
VL CDR3 amino acid sequence: QQRSNWPLT SEQ ID NO:486
Antibody STIM009
VH domain nucleotide sequence: 495 SEQ ID NO
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTGGTAGTACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATTAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGATTTTTACGATATTTTGACTGATAGTCCGTACTTCTACTACGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA
VH domain amino acid sequence: 494 of SEQ ID NO
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQINSLRAEDTAVYYCARDFYDILTDSPYFYYGVDVWGQGTTVTVSS
VH CDR1 amino acid sequence: GFTFSDYY SEQ ID NO:491
VH CDR2 amino acid sequence: ISSSGSTI SEQ ID NO:492
VH CDR3 amino acid sequence: ARDFYDILTDSPYFYYGVDV SEQ ID NO:493
VL domain nucleotide sequence: 502 of SEQ ID NO
GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA
VL domain amino acid sequence: 501 SEQ ID NO
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKVEIK
VL CDR1 amino acid sequence: QSLLHSNGYNY SEQ ID NO:498
VL CDR2 amino acid sequence: LGS SEQ ID NO 499
VL CDR3 amino acid sequence: MQALQTPRT SEQ ID NO:500
TABLE S1.SEQ ID NOS:1-342
TABLE S2.SEQ ID NOS:343-538
TABLE S3.SEQ ID NOS 539-562
Table S4: sequences of antibody heavy chain variable regions obtained from additional clonal lines
CDRs are defined according to IMGT.
Table S5: sequences of antibody light chain variable regions obtained from additional clonal lines
The N-terminal E and 5' nucleotide additions in CL-71642 are shown in bold. These additions did not recover upon sequencing but were determined to be present in the sequence, as compared to the relevant clonal lines as shown in figure 36. CDRs are defined according to IMGT.