Summary of The Invention
Traditional CAR-T cells targeting BCMA still face toxicity and relapse problems in treating MM. A meta-study showed that CRS and NTs occurred at 74% (95% CI, 56-91%) and 34% (95% CI, 24-43%) after receiving traditional CAR-T cell therapy targeting BCMA, with more than 3 levels of event occurrence at 25% (95% CI, 7-43%) and 12% (95% CI, 4-20%), respectively. In addition, the incidence of humoral immunity damage due to plasma cell depletion in the treated patients increases, 69% of 128 patients with Relapsed Refractory Multiple Myeloma (RRMM) treated with idecel have infections, and the incidence of infections is 22% above grade 3, most patients need anti-infection, leukopoiesis factor and immunoglobulin replacement therapy. Also, in 97 RRMM patients receiving Cilta-cel treatment, 56% and 20% developed infections and grade 3 or more. These clinical results indicate that there is a need to develop a regulatable BCMA-targeted CAR-T cell therapy.
The present inventors designed, constructed, based on the P329G mod-uCAR-T cell technology, P329G CAR-T cells targeting BCMA by binding to the Fc domain of an antibody that specifically binds BCMA molecules comprising the P329G mutation by research, and validated their specific, controllable anti-tumor effects in vitro and in vivo. Unlike traditional BCMA-targeted CAR-T cells that directly recognize killer MM cells, the pharmaceutical combination of the present invention comprises two components: BCMA-specific P329G antibodies and P329GCAR-T cells. After BCMA-specific P329G antibodies recognize BCMA-expressing tumor cells, i.e., P329G antibodies bridge P329G CAR-T cells to tumor cells as an aptamer molecule, P329G CAR-T cells redirect to tumor cells by recognizing the Fc domain of P329G antibodies, producing target-specific tumor recognition and killing effects (see fig. 1B). In the therapeutic methods using the pharmaceutical combinations of the invention, the P329G antibody acts as a bridge connecting P329G CAR-T cells and tumor cells, exerting a "molecular switch" effect, modulating P329G CAR-T cell activity.
The CAR-T cell product developed by the technology has the following remarkable characteristics: 1. by adjusting the administration frequency, dosage, route and function of the antibody, the acute and chronic toxic and side effects generated by the traditional CAR-T cell treatment are effectively avoided, the continuous expansion and survival of CAR-T cells in vivo are promoted, and the durable anti-tumor effect is achieved; 2. by combining and sequential antibody administration, clinical pain points such as primary tumor drug resistance, relapse after treatment and the like are effectively overcome on the premise of not changing CAR-T cells; 3. by combining antibodies aiming at different tumor antigens, the aim of treating different tumors is fulfilled on the premise of not changing CAR-T cells. Due to the maturation of the antibody drug market and lower cost, the therapy would be beneficial to reduce the cost of treatment and increase the accessibility of CAR-T cell therapies.
Thus, in a first aspect, the invention provides a P329G mutant antibody capable of specifically binding to a BCMA molecule, including, but not limited to ADI-38497 PG Ab (also referred to herein as "ADI-38497 PG antibody", "38497 PG Ab", "ADI-38497 PG IgG", "38497 PG IgG", "PG 38497 antibody"), as a "molecular switch" of an immune effector cell (e.g., T cell, NK cell) expressing a CAR polypeptide.
In some embodiments, the invention provides an antibody or antigen binding fragment that specifically binds to a BCMA molecule comprising a heavy chain variable region and a light chain variable region, wherein:
The heavy chain variable region comprises a CDR H1 as set forth in amino acid sequence SSSYYWT (SEQ ID NO: 25) according to Kabat numbering, or a variant of said CDR H1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; CDR H2 represented by amino acid sequence SISIAGSTYYNPSLKS (SEQ ID NO: 26), or a variant of said CDR H2 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid; and amino acid sequence DRGDQILDV (SEQ ID NO: 27), or a variant of said CDR H3 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid; the light chain variable region comprises a CDR L1 as set forth in amino acid sequence RASQSISRYLN (SEQ ID NO: 28) according to Kabat numbering, or a variant of said CDR L1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; CDR L2 represented by amino acid sequence AASSLQS (SEQ ID NO: 29), or a variant of said CDR L2 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid; and amino acid sequence QQKYFDIT (SEQ ID NO: 30), or a variant of said CDR L3 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid; wherein the amino acid change is an addition, deletion or substitution of an amino acid.
In some embodiments, the invention provides an antibody or antigen binding fragment that specifically binds to a BCMA molecule comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR H1 shown according to amino acid sequence SSSYYWT (SEQ ID NO: 25) of Kabat numbering; CDR H2 shown in amino acid sequence SISIAGSTYYNPSLKS (SEQ ID NO: 26); and CDR H3 shown in amino acid sequence DRGDQILDV (SEQ ID NO: 27); the light chain variable region comprises CDR L1 as shown in amino acid sequence RASQSISRYLN (SEQ ID NO: 28) according to Kabat numbering; CDR L2 represented by amino acid sequence AASSLQS (SEQ ID NO: 29); and CDR L3 shown in amino acid sequence QQKYFDIT (SEQ ID NO: 30).
In some embodiments, the invention provides an antibody or antigen binding fragment that specifically binds to a BCMA molecule comprising a heavy chain variable region comprising the sequence of SEQ ID No. 2 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.
In some embodiments, the invention provides an antibody or antigen binding fragment that specifically binds to a BCMA molecule comprising a light chain variable region comprising the sequence of SEQ ID NO:3 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.
In some embodiments, the invention provides an antibody or antigen binding fragment that specifically binds to a BCMA molecule comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence of SEQ ID No. 2 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and the light chain variable region comprises the sequence of SEQ ID NO. 3 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.
In some embodiments, the invention provides an antibody or antigen binding fragment that specifically binds to a BCMA molecule comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence of SEQ ID No. 2 and the light chain variable region comprises the sequence of SEQ ID No. 3.
In some embodiments, the antibody that specifically binds to a BCMA molecule of the present invention is an IgG1, igG2, igG3, or IgG4 antibody; preferably, it is an IgG1 or IgG4 antibody; more preferably, it is an IgG1 antibody.
In some embodiments, the antigen binding fragment of an antibody of the invention that specifically binds to a BCMA molecule is Fab, fab ', F (ab')2, fv, single chain Fab, diabody (diabody).
For the antibody or antigen binding fragment that specifically binds BCMA molecule, an antibody having a mutant Fc domain is obtained by mutating the amino acid at position P329 according to EU numbering to glycine (G), wherein the fcγ receptor binding of the mutant Fc domain is reduced compared to the fcγ receptor binding of the unmutated parent antibody Fc domain.
In some embodiments, the mutant Fc domain is a mutant Fc domain of an IgG1, igG2, igG3, or IgG4 antibody, preferably the mutant Fc domain is a mutant Fc domain of an IgG1 or IgG4 antibody; more preferably, the mutant Fc domain is a mutant Fc domain of an IgG1 antibody;
In some embodiments, an antibody or antigen binding fragment that specifically binds a BCMA molecule comprises the heavy chain constant region sequence shown in SEQ ID No. 5 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto and wherein the amino acid at position P329 according to EU numbering is mutated to G.
In some embodiments, an antibody or antigen binding fragment that specifically binds a BCMA molecule comprises the light chain constant region sequence shown in SEQ ID No. 6 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.
In some embodiments, an antibody or antigen binding fragment that specifically binds a BCMA molecule comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region comprises the amino acid sequence shown in SEQ ID No. 5 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto and wherein the amino acid at position P329 according to EU numbering is mutated to G; and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO. 6 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.
In one embodiment, an antibody or antigen binding fragment that specifically binds to a BCMA molecule comprises the heavy chain constant region sequence shown in SEQ ID No. 5 and the light chain constant region sequence shown in SEQ ID No. 6.
In some embodiments, the invention provides an antibody or antigen binding fragment that specifically binds to a BCMA molecule comprising a heavy chain, wherein the heavy chain comprises the sequence of SEQ ID No. 34 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto and wherein the amino acid at position P329 according to EU numbering is mutated to G.
In some embodiments, the invention provides an antibody or antigen binding fragment that specifically binds to a BCMA molecule comprising a light chain, wherein the light chain comprises the sequence of SEQ ID No. 35 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.
In some embodiments, the invention provides an antibody or antigen binding fragment that specifically binds to a BCMA molecule comprising a heavy chain and a light chain, wherein the heavy chain comprises the sequence of SEQ ID No. 34 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto and wherein the amino acid at position P329 according to EU numbering is mutated to G; and the light chain comprises the sequence of SEQ ID NO. 35 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.
In some embodiments, the invention provides an antibody or antigen binding fragment that specifically binds to a BCMA molecule comprising a heavy chain and a light chain, wherein the heavy chain comprises the sequence of SEQ ID No. 34 and the light chain comprises the sequence of SEQ ID No. 35.
In a second aspect, the invention provides a nucleic acid encoding an antibody of the first aspect of the invention, a vector comprising a nucleic acid encoding said antibody, a cell comprising said nucleic acid molecule or vector, and a method of making said antibody, said method comprising culturing a host cell into which an expression vector encoding a nucleic acid encoding an antibody or antigen binding fragment of a BCMA molecule specifically binding to BCMA molecule of the first aspect is introduced under conditions suitable for expression of a nucleic acid encoding said antibody or antigen binding fragment of a BCMA molecule specifically binding to the first aspect, isolating said antibody or antigen binding fragment of a BCMA molecule specifically binding to BCMA, optionally said method further comprising recovering said antibody or antigen binding fragment of a BCMA molecule from said host cell. Preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from the group consisting of E.coli cells, yeast cells, mammalian cells or other cells suitable for the preparation of antibodies or antigen-binding fragments thereof, most preferably the host cell is a HEK293 cell or a CHO cell.
In a third aspect, the present invention provides a pharmaceutical combination comprising
(I) A first component selected from the group consisting of immune effector cells (e.g., T cells, NK cells) expressing a molecular switch-regulated CAR polypeptide, a nucleic acid molecule encoding the CAR polypeptide, a vector comprising the nucleic acid molecule, and any combination thereof; and
(Ii) A second component which is an antibody or antigen-binding fragment that specifically binds to a BCMA molecule comprising a P329G mutation (also referred to as a P329G mutant antibody), e.g., a P329G mutant antibody of the first aspect of the invention, and
Optionally pharmaceutically acceptable excipients;
wherein the molecular switch regulated CAR polypeptide comprises
(1) A humanized anti-P329G mutant scFv sequence, wherein the scFv sequence comprises the following sequence capable of specifically binding to an antibody Fc domain comprising a P329G mutation, but not to an unmutated parent antibody Fc domain:
(i) A heavy chain variable region comprising a sequence numbered according to Kabat
(A) A heavy chain complementarity determining region CDR H1 represented by amino acid sequence RYWMN (SEQ ID NO: 19), or a variant of said CDR H1 having NO more than 2 amino acid changes or NO more than 1 amino acid change;
(b) CDR H2 represented by amino acid sequence EITPDSSTINYAPSLKG (SEQ ID NO: 20), or a variant of said CDR H2 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid; and
(C) A CDR H3 represented by amino acid sequence PYDYGAWFAS (SEQ ID NO: 21), or a variant of said CDR H3 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid; and
(Ii) A light chain variable region comprising a sequence numbered according to Kabat
(D) A light chain complementarity determining region (CDR L) 1 represented by amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 22), or a variant of said CDR L1 having NO more than 2 amino acid changes or NO more than 1 amino acid change;
(e) CDR L2 represented by amino acid sequence GTNKRAP (SEQ ID NO: 23), or a variant of said CDR L2 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid; and
(F) A CDR L3 represented by amino acid sequence ALWYSNHWV (SEQ ID NO: 24), or a variant of said CDR L3 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid;
Wherein the amino acid change is an addition, deletion or substitution of an amino acid;
(2) A hinge/spacer region selected from
(I) (G4S)n、(SG4)n or G4(SG4)n peptide linker, wherein "n" is an integer from 1 to 10, such as an integer from 1 to 4; such as the sequence shown in SEQ ID NO: 14;
(ii) A CD8 alpha hinge region or a variant thereof having 1-5 amino acid modifications, for example, the sequence shown in SEQ ID NO. 18 or a variant thereof having 1-2 amino acid modifications;
(3) A transmembrane region (TM) selected from the CD8 transmembrane domain or a variant thereof having 1-5 amino acid modifications, for example the sequence shown in SEQ ID NO. 15 or a variant thereof having 1-2 amino acid modifications;
(4) A Costimulatory Signal Domain (CSD) selected from the group consisting of a 4-1BB costimulatory domain or a variant thereof having 1-5 amino acid modifications, e.g.the sequence shown in SEQ ID NO. 16 or a variant thereof having 1-2 amino acid modifications;
(5) A Stimulatory Signaling Domain (SSD) being a CD3 delta signaling domain or a variant thereof having 1-10 amino acid modifications, e.g., the sequence shown in SEQ ID NO. 17 or a variant thereof having 1-10, 1-5 amino acid modifications;
Wherein the amino acid modification is an addition, deletion or substitution of an amino acid.
In some embodiments, the molecular switch-regulated CAR polypeptide described in the pharmaceutical combination of the invention comprises
(1) A humanized anti-P329G mutant scFv sequence, wherein the scFv sequence comprises the following sequence capable of specifically binding to an antibody Fc domain comprising a P329G mutation, but not to an unmutated parent antibody Fc domain:
(i) A heavy chain variable region comprising a sequence numbered according to Kabat
(A) CDR H1 shown in amino acid sequence RYWMN (SEQ ID NO: 19);
(b) CDR H2 represented by amino acid sequence EITPDSSTINYAPSLKG (SEQ ID NO: 20); and
(C) CDR H3 shown in amino acid sequence PYDYGAWFAS (SEQ ID NO: 21); and
(Ii) A light chain variable region comprising a sequence numbered according to Kabat
(D) CDR L1 represented by amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 22);
(e) CDR L2 represented by amino acid sequence GTNKRAP (SEQ ID NO: 23); and
(F) CDR L3 represented by amino acid sequence ALWYSNHWV (SEQ ID NO: 24);
For example, (i) a heavy chain variable region comprising the sequence of SEQ ID NO. 12 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and
(Ii) A light chain variable region comprising the sequence of SEQ ID No. 13 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
For example, (i) a heavy chain variable region comprising the sequence of SEQ ID NO. 12, and (ii) a light chain variable region comprising the sequence of SEQ ID NO. 13;
(2) A hinge/spacer region selected from
(I) (G4S)n、(SG4)n or G4(SG4)n peptide linker, wherein "n" is an integer from 1 to 4, such as the sequence shown in SEQ ID NO: 14;
(ii) A CD 8a hinge region sequence shown in SEQ ID No. 18 or a variant thereof having 1 amino acid modification;
(3) A transmembrane region (TM) selected from the CD8 transmembrane domain shown in SEQ ID NO. 15 or a variant thereof having 1 amino acid modification;
(4) A Costimulatory Signaling Domain (CSD) selected from the 4-1BB costimulatory domain shown in SEQ ID NO. 16 or a variant thereof with 1 amino acid modification;
(5) A Stimulatory Signaling Domain (SSD) selected from the group consisting of the CD3δ signaling domain shown in SEQ ID NO. 17 or a variant thereof having 1 amino acid modification;
Wherein the amino acid modification is an addition, deletion or substitution of an amino acid.
In some embodiments, the molecular switch regulated CAR polypeptides described in the pharmaceutical combinations of the invention further comprise a signal peptide sequence at the N-terminus, e.g., the signal peptide sequence shown in SEQ ID NO. 11,
In some embodiments, the molecular switch regulated CAR polypeptide in the pharmaceutical combination of the invention has the amino acid sequence shown in SEQ ID No. 1 or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.
In a fourth aspect, the invention provides a nucleic acid encoding a molecular switch regulated CAR polypeptide as described in the pharmaceutical combination of the invention, a vector comprising a nucleic acid encoding the CAR polypeptide, and a cell comprising the CAR nucleic acid molecule or vector, or a cell expressing the CAR polypeptide, preferably the cell is an autologous T cell or an allogeneic T cell.
In some embodiments, the nucleic acid molecule encoding a molecular switch regulated CAR polypeptide described in the pharmaceutical combination of the invention is a nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID No. 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.
In some embodiments, the vector comprising a nucleic acid molecule encoding a molecular switch-regulated CAR polypeptide described in a pharmaceutical combination of the invention is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector.
In some embodiments, the immune effector cells in the pharmaceutical combination of the invention are autologous T cells, NK cells or allogeneic T cells, NK cells prepared from T cells, NK cells expressing the molecular switch-regulated CAR polypeptides of the invention, e.g., T cells isolated from human peripheral blood mononuclear cells (PERIPHERAL BLOOD MONONUCLEAR CELL, PBMCs), NK cells prepared from T cells, NK cells expressing the molecular switch-regulated CAR polypeptides of the invention.
In some embodiments, the invention utilizes primary P329GCAR-T cells prepared from human PBMCs from a plurality of different donor sources, and utilizes P329G mutated anti-BCMA humanized antibodies, to evaluate the effect function of P329GCAR-T cells on BCMA expressing tumor cells by antibodies in an in vitro co-culture system, whereby P329G mutated anti-BCMA antibodies can act as a "molecular switch" to modulate the recognition and killing activity of P329G CAR-T cells on BCMA positive tumor cells, which is comparable to conventional CAR-T cells that directly target BCMA positive tumor cells, but the activity of conventional CAR-T cells is independent of P329G mutated antibodies.
In yet other embodiments, the invention demonstrates the anti-tumor effect of P329G CAR-T cells in combination with P329G mutant antibodies in immunodeficient mice vaccinated with BCMA expression positive human tumor cell line-derived tumors, and studies are performed on antibody dosing doses, intervals, and the like.
In vitro and in vivo experiments show that the activity of the P329G CAR-T cells can be turned on only in the presence of the P329G antibody, so as to generate an effector function, and identify and kill BCMA positive tumor cells; by adjusting the dosage and the dosing interval of the P329G antibody, the in vivo expansion degree of the P329G CAR-T cells and the anti-tumor activity intensity of the P329G CAR-T cells can be regulated. Given that BCMA transmits a pro-tumor signal after binding to its ligand, the P329G antibody is able to exert an anti-tumor effect by blocking this signal, and thus BCMA-specific P329G antibodies and P329G CAR-T cells are also able to exert a synergistic anti-tumor effect.
In some embodiments, for the pharmaceutical combination of the invention, (i) immune effector cells expressing a CAR polypeptide of the invention are administered to the subject intravenously in a single or multiple times at about 0.5×106 cells/kg body weight to about 5×106 cells/kg body weight, e.g., about 0.75×106 cells/kg body weight, 1×106 cells/kg body weight, 1.5×106 cells/kg body weight, 2×106 cells/kg body weight, 2.5×106 cells/kg body weight, 3×106 cells/kg body weight, 3.5×106 cells/kg body weight, 4×106 cells/kg body weight, 4.5×106 cells/kg body weight, 5×106 cells/kg body weight; and
(Ii) The P329G mutant antibody described in the pharmaceutical combination of the invention is administered to the subject in the form of a dosage unit of about 0.1-3mg/kg, preferably about 0.1mg/kg, 0.2mg/kg, 0.3mg/kg, 0.4mg/kg, 0.5mg/kg, 1mg/kg, 1.5mg/kg, 2mg/kg, 2.5mg/kg, 3mg/kg, preferably parenterally, more preferably intravenously.
In some embodiments, (i) and (ii) in the pharmaceutical combination of the invention are administered separately, simultaneously or sequentially, e.g., on the first day, (ii), intravenously on the same day or the second day, and then multiple administrations (ii) at a frequency.
In some preferred embodiments, the (i) and (ii) of the pharmaceutical combination of the invention are administered according to the following schedule:
Sequentially administering (ii) and (i) on a first day, at intervals of about 1-5 hours (e.g., about 1, 2, 3, 4, or 5 hours apart), preferably, administering (ii) on a first day, at intervals of about 1-5 hours (e.g., about 1, 2, 3, 4, or 5 hours apart); or (ii) on the first day, and (i) on the second day, at intervals of about 24 hours;
Taking the first day as the starting day, the administration (ii) is performed every 1 week (QW), 2 weeks (Q2W) or 3 weeks (Q3W) in the first period; optionally, a plurality of
Second to nth cycles: every three weeks (Q3W) of administration (ii) in each cycle.
In some embodiments, each dosing cycle is 21 days or 28 days.
In some embodiments, at least 2-3 cycles of administration. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cycles or more are administered.
In a fourth aspect, the present invention provides a pharmaceutical combination of the invention or use thereof for the treatment of a BCMA-related disease in a subject comprising administering to the subject a therapeutically effective amount of a pharmaceutical combination as defined in the foregoing third aspect, wherein the BCMA-related disease is, for example, a BCMA expressing or overexpressing cancer, for example, multiple myeloma, for example refractory multiple myeloma or relapsed multiple myeloma, for example, the multiple myeloma is relapsed/refractory multiple myeloma (relapsed/refractory multiple myeloma, RRMM).
In a fifth aspect, the invention provides the use of a pharmaceutical combination of the invention in the manufacture of a medicament for the treatment of a disease associated with BCMA, e.g. a BCMA expressing or overexpressing cancer, e.g. multiple myeloma, e.g. refractory multiple myeloma or relapsed multiple myeloma, e.g. relapsed/refractory multiple myeloma (relapsed/refractory multiple myeloma, RRMM).
In a sixth aspect, the invention provides a method for treating a disease associated with BCMA, comprising administering to a subject a therapeutically effective amount of a pharmaceutical combination of the invention, e.g. a BCMA expressing or overexpressing cancer, e.g. multiple myeloma, e.g. refractory multiple myeloma or relapsed multiple myeloma, e.g. the multiple myeloma is relapsed/refractory multiple myeloma (relapsed/refractory multiple myeloma, RRMM).
In a seventh aspect, the present invention provides a kit comprising a pharmaceutical combination as defined in the preceding third aspect, preferably the kit is in the form of a pharmaceutical dosage unit.
Thus, in a first aspect of the invention, by in vitro binding capacity, affinity and Fc effect functional assays, a high affinity BCMA-specific P329G antibody is obtained which is capable of binding both BCMA antigen and P329G CAR molecules and exerting a bridging effect. Second, the present invention in a third aspect constructs an in vitro co-culture system by using a constructed P329G CAR structural molecule and combining P329G CAR-T cells prepared from the CAR molecule with BCMA specific P329G antibodies and then co-culturing with BCMA positive MM cells in vitro, in which system the effect of the P329G antibodies as a "molecular switch" to modulate the activity of the P329G CAR-T cells was verified, i.e. only in the presence of P329G mutant antibodies, P329G CAR-T cells could be activated, proliferated, secrete effector cytokines and produce killing effects, and these effects exhibited P329G antibody dose dependence, with increasing antibody doses, P329G CAR-T cell recognition and killing effects. Whereas WT antibodies that do not carry the P329G mutation are unable to elicit P329G CAR-T cell effector function.
In addition, in vitro experiments of the present invention demonstrate that soluble BCMA antigen does not affect P329G CAR-T cell activity when used in combination with BCMA specific P329G antibodies, whereas soluble BCMA antigen produces a significant inhibitory effect on conventional CAR-T cells. In the MM cell subcutaneous and systemic tumor-bearing immunodeficiency mouse models with high and low BCMA expression, the P329G CAR-T cell combined with the P329G antibody generates good anti-tumor effect which is at least equivalent to that of the traditional CAR-T cell, but the in vivo expansion degree of the P329G CAR-T cell is obviously lower than that of the traditional CAR-T cell, which indicates that the P329G CAR-T cell possibly induces lower acute toxic and side effects such as CRS, NT and the like while generating equivalent anti-tumor effect; furthermore, the in vivo expansion degree and the anti-tumor effect intensity of the P329G CAR-T cells can be regulated by regulating the dosage and the interval of the P329G antibody administration, and after tumor elimination, the P329G antibody administration is stopped, and the sustained anti-tumor effect can still be maintained, so that a foundation is laid for 'closing' the activity of the P329G CAR-T cells in clinical application, maintaining the sustained anti-tumor effect, and simultaneously recovering the normal plasma cell quantity, reducing the humoral immunity, and other long-term toxic and side effects.
The toxicological experiment of the invention also shows that the P329G CAR-T cell combined P329G antibody does not generate obvious toxicity, thereby laying a foundation for clinical transformation.
In some embodiments, the subject has a low probability of developing CRS or ICANS, e.g., less than 50%, e.g., less than an existing BCMA-CAR-T product, following administration of the therapy of the present invention.
In some embodiments, the therapies of the invention have good safety and/or tolerability.
In some embodiments, the second component (e.g., an anti-BCMA antibody) is capable of modulating the activity of the first component, particularly the cellular activity of the CAR-T cells.
In some embodiments, the therapies of the invention are capable of partially alleviating a disease in a subject, and clinical studies have shown that a panel of 11 patients are pooled, with 5 patients achieving PR and above remission.
In some specific embodiments, the pharmaceutical combination or therapy of the invention achieves a better effect on safety (1) CRS: by the date of data expiration, 5 out of 11 subjects developed different degrees of CRS with a 45.5% incidence of CRS, lower than existing BCMA-CART products, in which case the CRS that had developed was relatively controllable. Most subjects had improved or healed after treatment;
(2) ICANS: by the date of data expiration, both studies were performed and no neurological toxic events were seen.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
I. definition of the definition
For purposes of explaining the present specification, the following definitions will be used, and terms used in the singular form may also include the plural, and vice versa, as appropriate. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
The term "about" when used in conjunction with a numerical value is intended to encompass numerical values within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value.
As used herein, the term "and/or" means any one of the selectable items or two or more of the selectable items.
In this document, the terms "comprises" or "comprising" when used herein, unless otherwise indicated, also encompass the circumstance of consisting of the recited elements, integers or steps. For example, when referring to an antibody variable region "comprising" a particular sequence, it is also intended to encompass antibody variable regions consisting of that particular sequence.
The terms "BCMA" and "B cell maturation antigen" are used interchangeably and include variants, isoforms, species homologs, and analogs of human BCMA having at least one identical epitope as BCMA (e.g., human BCMA). BCMA proteins may also include fragments of BCMA, such as extracellular domains and fragments of extracellular domains, e.g., fragments that retain the binding ability to any antibody of the invention.
The terms "BCMA antibody", "antibody against BCMA", "antibody specifically binding to BCMA", "antibody specifically targeting BCMA", "antibody specifically recognizing BCMA" as used herein are used interchangeably to mean an antibody capable of specifically binding to B Cell Maturation Antigen (BCMA).
The term "antibody" is used herein in its broadest sense to refer to a protein comprising an antigen binding site, and encompasses natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single chain antibodies, intact antibodies, and antibody fragments. Preferably, the antibodies of the invention are single domain antibodies or heavy chain antibodies.
An "antibody fragment" or "antigen-binding fragment" is used interchangeably herein to refer to a molecule that is different from an intact antibody, which comprises a portion of the intact antibody and binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, fab ', F (ab') 2, fv, single chain Fab, diabody (diabody).
The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are continuously linked, optionally via a flexible short polypeptide linker, and are capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. As used herein, an scFv may have a VL variable region and a VH variable region in any order (e.g., with respect to the N-terminus and C-terminus of the polypeptide), unless otherwise indicated, an scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL.
"Complementarity determining regions" or "CDR regions" or "CDRs" or "hypervariable regions" are regions of an antibody variable domain that are hypervariable in sequence and form structurally defined loops ("hypervariable loops") and/or contain antigen-contacting residues ("antigen-contacting points"). CDRs are mainly responsible for binding to the epitope. CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. CDRs located within the antibody heavy chain variable domain are referred to as CDR H1, CDR H2 and CDR H3, while CDRs located within the antibody light chain variable domain are referred to as CDR L1, CDR L2 and CDR L3. In a given light chain variable region or heavy chain variable region amino acid sequence, the exact amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example: chothia (Chothia et al, (1989) Nature 342:877-883), al-Lazikani et al ,"Standard conformations for the canonical structures of immunoglobulins",Journal of Molecular Biology,273,927-948(1997)), are based on Kabat (Kabat et al, sequences of Proteins of Immunological Interest, 4 th edition ,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath),Contact(University College London), International ImMunoGeneTics database (IMGT) (world Wide Web IMGT. Circuits. Fr /) of antibody sequence variability, and North CDR definitions based on neighbor-propagating clusters (affinity propagation clustering) using a large number of crystal structures.
In the present invention, unless otherwise indicated, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-described ways.
CDRs may also be determined based on having the same Kabat numbering positions as the reference CDR sequences (e.g., any of the CDRs of the examples of the invention). In the present invention, when referring to the antibody variable region and specific CDR sequences (including heavy chain variable region residues), reference is made to numbering positions according to the Kabat numbering system.
Although CDRs vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. Using at least two of the Kabat, chothia, abM and Contact methods, the minimum overlap region can be determined, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit may be a sub-portion of the CDR. As will be apparent to those skilled in the art, the residues in the remainder of the CDR sequences can be determined by the structure of the antibody and the protein folding. Thus, the present invention also contemplates variants of any of the CDRs presented herein. For example, in a variant of one CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia or AbM may be replaced by conserved amino acid residues.
The term "chimeric antibody" is an antibody molecule in which (a) a constant region or portion thereof is altered, substituted, or exchanged such that the antigen binding site is linked to a constant region of a different or altered class and/or species or an entirely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug) or the like that confers novel properties to the chimeric antibody; or (b) altering, replacing or exchanging the variable region or a portion thereof with a variable region having a different or altered antigen specificity. For example, a murine antibody may be modified by replacing its constant region with a constant region derived from a human immunoglobulin. Due to the replacement with human constant regions, the chimeric antibody can retain its specificity in recognizing an antigen while having reduced antigenicity in humans as compared to the original murine antibody.
"Humanized" antibody refers to a chimeric antibody comprising amino acid residues from a non-human CDR and amino acid residues from a human FR. In some embodiments, all or substantially all CDRs (e.g., CDRs) in a humanized antibody correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.
"Human antibody" refers to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source that utilizes a human antibody repertoire or other human antibody coding sequence. This definition of human antibodies specifically excludes humanized antibodies that comprise non-human antigen binding residues.
The term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. The human IgG heavy chain Fc region is generally defined as the region from the amino acid residue at position Cys226 or Pro230 to the carboxy terminus thereof, and the lysine residue at position 447 of the C-terminus of the Fc region (according to the EU numbering system) may be present or absent. Thus, the whole antibody composition may include a population of antibodies in which all of the K447 residues are deleted, a population of antibodies in which no K447 residues are deleted, or a population of antibodies that combine an antibody having a K447 residue with an antibody having no K447 residue.
In certain embodiments, the Fc region of the immunoglobulin comprises two constant domain domains, i.e., CH2 and CH3, and in other embodiments, the Fc region of the immunoglobulin comprises three constant domains, i.e., CH2, CH3, and CH4.
Binding of IgG to fcγ receptor or C1q depends on residues located in the hinge region and CH2 domains. The two regions of the CH2 domain are critical for fcγr and complement C1q binding and have unique sequences in IgG2 and IgG 4. Substitution of residues 233-236 in human IgG1 and IgG2 and substitution of residues 327, 330 and 331 in human IgG4 has been shown to significantly reduce ADCC and CDC activity (Armour et al, eur. J. Immunol.29 (8), 1999, 2613-2624; shields et al, J. Biol. Chem.276 (9), 2001, 6591-6604).
"Functional Fc region" and like terms are used interchangeably to refer to an Fc region having the effector function of a wild-type Fc region. In some embodiments, the Fc region comprises the amino acid sequence set forth in SEQ ID NO 9, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence.
The terms "variant Fc region," "Fc mutant," "Fc region carrying a mutation," "mutant Fc region," "variant Fc region," and "mutant Fc region" and the like are used interchangeably to refer to an Fc region comprising at least one amino acid modification that differs from the native sequence Fc region/wild-type Fc region.
In some embodiments, the variant Fc region comprises an amino acid sequence that differs from the amino acid sequence of the native sequence Fc region by one or more amino acid substitutions, deletions, or additions. In some embodiments, the variant Fc region has at least one amino acid substitution compared to the Fc region of a wild-type IgG, the at least one amino acid substitution being a substitution of an amino acid at position P329 according to EU numbering with glycine (G). In some embodiments, the Fc region comprises the amino acid sequence set forth in SEQ ID NO. 10, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence and has a P329G mutation.
"Fc receptor" or "FcR" refers to a molecule that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR is a receptor that binds an IgG antibody, i.e., fcγr, including three receptors, fcγri (CD 64), fcγrii (CD 32) and fcγriii (CD 16), as well as allelic variants and alternatively spliced forms of these receptors. Fcyrii receptors include fcyriia and fcyriib, and fcyriii receptors include fcyriiia and fcyriiib.
The term "effector functions" refers to those biological activities attributed to the Fc region of an immunoglobulin that vary with the immunoglobulin isotype. Examples of immunoglobulin effector functions include: fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, C1q binding and complement-dependent cytotoxicity (CDC), down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.
The term "antibody-dependent cell-mediated cytotoxicity (ADCC)" is one of the major mechanisms by which certain cytotoxic effector cells, such as Natural Killer (NK) cells, mediate killing of target cells and foreign host cells. In some embodiments, the chimeric antigen receptor of the invention provides antibody-dependent cellular cytotoxicity of T lymphocytes, enhancing antibody-dependent cellular cytotoxicity of NK cells. The chimeric antigen receptor of the present invention induces activation, sustained proliferation and development of specific cytotoxicity to a target cancer cell mediated through an antibody (or other anti-tumor molecule comprising an Fc portion) that binds to a tumor cell by binding to the antibody (or other anti-tumor molecule comprising an Fc portion) expressing the chimeric antigen receptor.
The term "antibody-dependent cellular phagocytosis (ADCP)" refers to a cellular response in which activation of macrophages is induced by binding of antibodies that bind to target cells to fcyriiia on the surface of the macrophages, thereby internalizing the target cells and degrading by phagosome acidification. ADCP may also be mediated by fcyriia and fcyri, but is relatively small in occupancy.
The term "Complement Dependent Cytotoxicity (CDC)" refers to the lysis of target cells in the presence of complement. The complement system is part of the innate immune system, which consists of a range of proteins. Proteins of the complement system, designated by the abbreviations C1, C2, C3, etc., are a group of thermolabile, enzymatically active proteins found in human or vertebrate serum, interstitial fluid. C1q is the first component of the complement-dependent cytotoxicity (CDC) pathway that is capable of binding six antibodies, but binding to two IgG is sufficient to activate the complement cascade. Activation of the classical complement pathway is initiated by binding of the first component of the complement system (C1 q) to an antibody (appropriate subclass) that binds to the relevant antigen, activating a series of complement cascades that form pores in the target cell membrane, resulting in target cell death. To assess complement activation, CDC assays may be performed, for example, by the method described in Gazzano-Santoro et al, J.Immunol. Methods 202:163 (1996).
The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies generally have similar structures, with each domain comprising four conserved Framework Regions (FR) and three Complementarity Determining Regions (CDRs). (see, e.g., kindt et al Kuby Immunology,6th ed., W.H. Freeman and Co.91 page (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity.
As used herein, the term "bind" or "specifically bind" means that the binding is selective for an antigen and distinguishable from unwanted or non-specific interactions. The ability of an antibody to bind to a particular antigen may be determined by enzyme-linked immunosorbent assay (ELISA), SPR or biofilm layer interference techniques or other conventional binding assays known in the art.
The term "stimulation" refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex) to its corresponding ligand, which thus mediates signaling events, such as, but not limited to, signaling via the TCR/CD3 complex. Stimulation may mediate the altered expression of certain molecules, such as down-regulation of TGF-beta and/or reorganization of cytoskeletal structures, and the like.
The term "stimulatory molecule" refers to a molecule expressed by a T cell that provides a primary cytoplasmic signaling sequence that modulates primary activation of the TCR complex in a stimulatory manner in at least some aspect of the T cell signaling pathway. In one embodiment, the primary signal initiates and results in mediating T cell responses including, but not limited to, proliferation, activation, differentiation, etc., e.g., through binding of the TCR/CD3 complex to peptide-loaded MHC molecules. In a particular CAR of the invention, the intracellular signaling domain in any one or more CARs of the invention comprises an intracellular signaling sequence, e.g., a primary signaling sequence of cd3δ.
The term "cd3δ" is defined as a protein provided by accession number GenBan BAG36664.1 or an equivalent thereof, and "cd3δ stimulatory signaling domain" is defined as an amino acid residue from the cytoplasmic domain of the cd3δ chain sufficient to functionally propagate the initial signal necessary for T cell activation. In one embodiment, the cytoplasmic domain of cd3δ comprises residues 52 to 164 of GenBank accession No. BAG36664.1 or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.) as functional orthologs thereof. In one embodiment, the "CD3δ stimulatory signaling domain" is the sequence provided in SEQ ID NO. 17 or a variant thereof.
The term "costimulatory molecule" refers to a corresponding binding partner on a cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response (such as, but not limited to, proliferation) of the cell. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an effective immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activation of NK cell receptors, OX40, CD40, GITR, 4-1BB (i.e., CD 137), CD27, and CD28. In some embodiments, the "costimulatory molecule" is 4-1BB (i.e., CD 137). The costimulatory signal domain refers to the intracellular portion of a costimulatory molecule.
The term "4-1BB" refers to a TNFR superfamily member having the amino acid sequence provided as GenBank accession No. AAA62478.2 or an equivalent residue from a non-human species (e.g., mouse, rodent, monkey, ape, etc.); and "4-1BB costimulatory signaling domain" is defined as amino acid residues 214-255 of GenBank accession AAA62478.2 or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). In one embodiment, a "4-1BB co-stimulatory domain" is a sequence provided as SEQ ID NO. 16 or an equivalent residue from a non-human species (e.g., mouse, rodent, monkey, ape, etc.).
The term "signaling pathway" refers to a biochemical relationship between a plurality of signaling molecules that function in propagating a signal from one portion of a cell to another portion of the cell.
The term "cytokine" is a generic term for proteins released by one cell population that act as intercellular mediators on another cell. Examples of such cytokines are lymphokines, monokines, interleukins (IL), such as IL-1, IL-1. Alpha., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; tumor necrosis factors such as TNF- α or TNF- β; and other polypeptide factors including gamma interferon.
An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibodies of the invention are purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For reviews of methods for assessing antibody purity, see, e.g., flatman et al, j. Chromatogr.b848:79-87 (2007).
An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. An "isolated nucleic acid encoding an antibody of the invention" refers to one or more nucleic acid molecules encoding a chain of an antibody of the invention, or a fragment thereof, including such nucleic acid molecules in a single vector or in separate vectors, as well as such nucleic acid molecules present at one or more locations in a host cell.
Calculation of sequence identity between sequences was performed as follows.
To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, the length of the reference sequences aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequences. Amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
Sequence comparison and calculation of percent identity between two sequences can be accomplished using mathematical algorithms. In a preferred embodiment, the percentage identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) J.mol.biol.48:444-453) algorithms (available at http:// www.gcg.com) that have been integrated into the GAP program of the GCG software package, using the Blossum 62 matrix or the PAM250 matrix and the GAP weights 16, 14, 12, 10, 8, 6 or 4 and the length weights 1,2, 3, 4, 5 or 6. In yet another preferred embodiment, the percentage of identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http:// www.gcg.com) using the NWS gapdna.CMP matrix and the GAP weights 40, 50, 60, 70 or 80 and the length weights 1,2, 3, 4, 5 or 6. A particularly preferred set of parameters (and one that should be used unless otherwise indicated) is the Blossum 62 scoring matrix employing gap penalty 12, gap extension penalty 4, and frameshift gap penalty 5.
The percent identity between two amino acid sequences or nucleotide sequences can also be determined using PAM120 weighted remainder table, gap length penalty 12, gap penalty 4) using the e.meyers and w.miller algorithm that has been incorporated into the ALIGN program (version 2.0) ((1989) CABIOS, 4:11-17).
Additionally or alternatively, the nucleic acid sequences and protein sequences described herein may be further used as "query sequences" to perform searches against public databases, for example, to identify other family member sequences or related sequences.
The terms "amino acid change" and "amino acid modification" are used interchangeably to refer to additions, deletions, substitutions and other modifications of amino acids. Any combination of amino acid additions, deletions, substitutions and other modifications may be made provided that the final polypeptide sequence has the desired properties. In some embodiments, amino acid substitutions to the antibody result in reduced binding of the antibody to the Fc receptor. For the purpose of altering the binding characteristics of, for example, an Fc region, non-conservative amino acid substitutions, i.e., substitution of one amino acid with another amino acid having a different structure and/or chemical nature, are particularly preferred. Amino acid substitutions include substitutions with non-naturally occurring amino acids or naturally occurring amino acid derivatives of the twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Genetic or chemical methods known in the art may be used to create amino acid changes. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, and the like. Methods of altering amino acid side chain groups by methods other than genetic engineering (e.g., chemical modification) may be useful. Various names may be used herein to represent identical amino acid changes. For example, the substitution of proline to glycine at position 329 of the Fc domain may be denoted 329G, G, G329, P329GPro329Gly, or simply "PG".
The terms "conservative sequence modifications", "conservative sequence changes" refer to amino acid modifications or changes that do not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing an amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications may be introduced to the antibodies or antibody fragments of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative substitutions are amino acid substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CAR of the invention may be replaced with other amino acid residues from the same side chain family, and the altered CAR may be tested using the functional assay described herein.
The term "autologous" refers to any substance that is derived from the same individual that will later reintroduce the substance to the individual.
The term "allogeneic" refers to any substance derived from a different animal of the same species as the individual into which the substance was introduced. Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical. In some aspects, the allografts from individuals of the same species may be sufficiently genetically dissimilar to occur antigenic interactions.
The term "xenogeneic" refers to grafts derived from animals of different species.
The term "apheresis" as used herein refers to an art-recognized in vitro method by which a donor or patient's blood is removed from the donor or patient and passed through a device that separates selected specific components and returns the remainder to the donor or patient's circulation, for example, by re-transfusion. Thus, in the context of "apheresis" reference is made to a sample obtained using apheresis.
The term "immune effector cell" refers to a cell that is involved in an immune response, e.g., involved in promoting an immune effector response. Examples of immune effector cells include T cells, e.g., alpha/beta T cells and gamma/delta T cells, B cells, natural Killer (NK) cells, natural Killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.
An "immune effector function", "immune effector response" or "immune effector response" refers to, for example, enhancement of an immune effector cell or promotion of a function or response of an immune attack target cell. For example, immune effector function or response refers to T cell or NK cell characteristics that promote killing or inhibit growth or proliferation of target cells. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.
The term "effector function" refers to a specialized function of a cell. The effector function of T cells may be, for example, cytolytic activity or helper activity, including secretion of cytokines.
The term "T cell activation" refers to one or more cellular responses of T lymphocytes, in particular cytotoxic T lymphocytes, selected from the group consisting of: proliferation, differentiation, cytokine secretion, cytotoxic effector release, cytotoxic activity and expression of activation markers. The chimeric antigen receptor of the invention is capable of inducing T cell activation. Suitable assays for measuring T cell activation are described in the examples and are known in the art.
The term "lentivirus" refers to a genus of the retrovirus family (Retroviridae). Lentiviruses are unique among retroviruses in being able to infect non-dividing cells; they can deliver significant amounts of genetic information to host cells, so they are one of the most efficient methods of gene delivery vehicles. HIV, SIV and FIV are all examples of lentiviruses.
The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome and includes, inter alia, a self-inactivating lentiviral vector as provided in Milone et al mol. Ther.17 (8): 1453-1464 (2009). Other examples of lentiviral vectors that may be used clinically include, for example, but are not limited to, those from Oxford BioMedicaGene delivery techniques, LENTIMAXTM vector systems from Lentigen, and the like. Non-clinical types of lentiviral vectors are also available and known to those skilled in the art.
The term "BCMA-related disease" refers to any condition caused, exacerbated, or otherwise associated with increased expression or activity of BCMA.
The terms "individual" or "subject" are used interchangeably and include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.
The terms "tumor" and "cancer" are used interchangeably herein to encompass solid tumors and liquid tumors.
The terms "cancer" and "cancerous" refer to physiological conditions in a mammal in which cell growth is not regulated.
The term "tumor" refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancerous" and "tumor" are not mutually exclusive when referred to herein.
"Tumor immune escape" refers to the process by which tumors evade immune recognition and clearance. Thus, as a therapeutic concept, tumor immunity is "treated" when such evasion is reduced, and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage and tumor elimination.
The term "half-maximal effective concentration (EC50)" refers to the concentration of a drug, antibody, or toxin that induces a 50% response between baseline and maximum after a particular exposure time.
The term "rayon light activated cell sorting" or "FACS" refers to a specific type of flow cytometry. It provides a method of sorting heterogeneous mixtures of biological cells into two or more containers one cell at a time according to the specific light scattering and rayon light characteristics of each cell (flowmetric. "Sorting Out Fluorescence ACTIVATED CELL Sorting". 2017-11-09). The apparatus for performing FACS is known to those skilled in the art and commercially available to the public. Examples of such instruments include FACS Star Plus, FACScan and FACSort instruments from Becton Dickinson (Foster City, calif.), epics C from Coulter Epics Division (Hialeah, FL) and MoFlo from Cytomation (Colorado Springs, colorado).
The term "pharmaceutically acceptable adjuvant" refers to diluents, adjuvants (e.g. Freund's adjuvant (complete and incomplete)), excipients, buffers or stabilizers etc. for administration with the active substance.
As used herein, "treating" refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desirable therapeutic effects include, but are not limited to, preventing occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or moderating the disease state, and alleviating or improving prognosis. In some embodiments, the antibody molecules of the invention are used to delay disease progression or to slow disease progression.
The term "effective amount" refers to an amount or dose of an antibody or composition of the invention that, upon administration to a patient in single or multiple doses, produces a desired effect in a patient in need of treatment or prophylaxis. The effective amount can be readily determined by the attending physician as a person skilled in the art by considering a number of factors: species such as mammals; body weight, age, and general health; specific diseases involved; the extent or severity of the disease; response of individual patients; specific antibodies administered; mode of administration; the bioavailability characteristics of the administration formulation; a selected dosing regimen; and the use of any concomitant therapy.
"Therapeutically effective amount" means an amount effective to achieve the desired therapeutic result at the desired dosage and for the desired period of time. The therapeutically effective amount of an antibody or antibody fragment or composition thereof may vary depending on a variety of factors such as the disease state, age, sex and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or detrimental effect of the antibody or antibody fragment or composition thereof is less than a therapeutically beneficial effect. The "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 50%, 60% or 70% and still more preferably by at least about 80% or 90% relative to an untreated subject. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system that predicts efficacy in human tumors.
The term "pharmaceutical combination" or "pharmaceutical combination product" refers to a non-fixed combination (product) or a fixed combination (product), including but not limited to a kit. The term "non-fixed combination" means that the active ingredients (e.g., (i) P329G CAR-T cells, and (ii) P329G mutant antibodies to BCMA) are administered to a subject simultaneously, without specific time constraints, or sequentially at the same or different time intervals, in separate entities, wherein such administration provides effective treatment in the subject. The term "fixed combination" refers to the combination of P329G mutant antibodies against BCMA and P329G CAR-T cells of the invention each being administered simultaneously to a patient in the form of a specific single dose. The term "non-fixed combination" means that the combination of a 329G mutant antibody against BCMA and a P329GCAR-T cell of the invention is administered to a patient as separate entities simultaneously, concurrently or sequentially, without specific dosage and time constraints, wherein such administration provides therapeutically effective levels of the pharmaceutical combination of the invention in the patient. In a preferred embodiment, the pharmaceutical combination is a non-fixed combination.
The term "vector" as used herein when referring to a nucleic acid refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures and that bind to the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors".
The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. The progeny may not be exactly identical in nucleic acid content to the parent cell, but may comprise the mutation. Included herein are mutant progeny selected or selected for the same function or biological activity in the initially transformed cells. Host cells are any type of cellular system that can be used to produce the antibody molecules of the invention, including eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells; and prokaryotic cells, e.g., E.coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissue or animal tissue.
"Subject/patient sample" refers to a collection of cells, tissue or body fluids obtained from a patient or subject. The source of the tissue or cell sample may be solid tissue, like an organ or tissue sample or biopsy or puncture sample from fresh, frozen and/or preserved; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time of gestation or development of a subject. Tissue samples may contain compounds that are not naturally intermixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. Examples of tumor samples herein include, but are not limited to, tumor biopsies, fine needle aspirates, broncholavages, pleural fluid (hydrothorax), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples such as formalin-fixed, paraffin-embedded tumor samples or frozen tumor samples.
When referring to a disease, the term "treating" refers to alleviating the disease (i.e., slowing or preventing or reducing the progression of the disease or at least one clinical symptom thereof), preventing or delaying the onset or progression or progress of the disease.
The molecular switch regulated Chimeric Antigen Receptor (CAR) of the present invention
The present invention relates to chimeric antigen receptor polypeptides capable of specifically binding to the mutant Fc domain of antibodies directed against BCMA molecules. In particular, the chimeric antigen receptor of the invention comprises a humanized anti-P329G mutant scFv sequence, and the scFv sequence is capable of specifically binding to an antibody Fc domain comprising a P329G mutation, but is not capable of specifically binding to an unmutated parent antibody Fc domain. The binding of the antibody Fc domain comprising the P329G mutation to an Fc receptor (e.g., fcγ receptor) is reduced compared to the binding of the non-mutated parent antibody Fc domain to the Fc receptor.
The recombinant CAR constructs of the invention comprise a sequence encoding a CAR, wherein the CAR comprises a humanized anti-P329G mutant scFv sequence that specifically binds to the P329G mutated antibody Fc domain.
In one embodiment, the scFv sequence in the CAR construct of the invention comprises the following sequence:
(i) A heavy chain variable region comprising a sequence numbered according to Kabat
(A) A heavy chain complementarity determining region CDR H1 represented by amino acid sequence RYWMN (SEQ ID NO: 19), or a variant of said CDR H1 having NO more than 2 amino acid changes or NO more than 1 amino acid change;
(b) CDR H2 represented by amino acid sequence EITPDSSTINYAPSLKG (SEQ ID NO: 20), or a variant of said CDR H2 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid; and
(C) A CDR H3 represented by amino acid sequence PYDYGAWFAS (SEQ ID NO: 21), or a variant of said CDR H3 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid; and
(Ii) A light chain variable region comprising a sequence numbered according to Kabat
(D) A light chain complementarity determining region (CDR L) 1 represented by amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 22), or a variant of said CDR L1 having NO more than 2 amino acid changes or NO more than 1 amino acid change;
(e) CDR L2 represented by amino acid sequence GTNKRAP (SEQ ID NO: 23), or a variant of said CDR L2 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid; and
(F) A CDR L3 represented by amino acid sequence ALWYSNHWV (SEQ ID NO: 24), or a variant of said CDR L3 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid;
Wherein the amino acid change is an addition, deletion or substitution of an amino acid.
Further, the scFv may be linked at the N-terminus to a signal peptide sequence, e.g., a signal peptide sequence as shown in SEQ ID NO. 11, and the scFv may be linked at the C-terminus to an optional hinge/spacer sequence as provided in SEQ ID NO. 14 or SEQ ID NO. 18, a transmembrane region as provided in SEQ ID NO. 15, a co-stimulatory signal domain as shown in SEQ ID NO. 16, and an intracellular stimulatory signal domain comprising SEQ ID NO. 17 or a variant thereof, e.g., wherein the respective domains are contiguous with each other and in the same open reading frame to form a single fusion protein.
In some embodiments, the scFv domain comprises (i) a heavy chain variable region comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence of SEQ ID NO. 12, and (ii) a light chain variable region comprising or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence of SEQ ID NO. 13;
In some embodiments, the scFv domain comprises (i) a heavy chain variable region shown as SEQ ID NO. 12 and (ii) a light chain variable region shown as SEQ ID NO. 13. In one embodiment, the scFv domain further comprises a (Gly 4-Ser) n linker, wherein n is 1, 2, 3, 4, 5 or 6, preferably 3 or 4. The light chain variable region and the heavy chain variable region of the scFv may, for example, be in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.
In some embodiments, an exemplary CAR construct of the invention comprises a signal peptide sequence, a humanized anti-P329G mutant scFv sequence, a hinge/spacer region, a transmembrane domain, an intracellular co-stimulatory signaling domain, and an intracellular stimulatory signaling domain.
In one embodiment, the amino acid sequence of the full length CAR polypeptide is provided as SEQ ID NO. 1, as shown in the sequence Listing.
In some embodiments, the invention provides recombinant nucleic acid constructs comprising a nucleic acid molecule encoding a CAR of the invention, e.g., comprising a nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID No. 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 1. The CAR constructs encoding the invention can be obtained using recombinant methods well known in the art. Alternatively, the nucleic acid of interest may be produced synthetically, rather than by genetic recombination methods.
The invention includes retroviral vector constructs and lentiviral vector constructs that express CARs that can be directly transduced into cells.
In some embodiments, the nucleic acid sequence of the CAR construct of the invention is cloned into a lentiviral vector to produce a full length CAR construct in a single coding frame, and used for expression with the EF 1a promoter.
One of ordinary skill in the art will appreciate that CAR polypeptides of the invention can also be modified to vary in amino acid sequence, but not in the desired activity. For example, additional nucleotide substitutions can be made to the CAR polypeptide that result in amino acid substitutions at "non-essential" amino acid residues. For example, a non-essential amino acid residue in a molecule may be replaced with another amino acid residue from the same side chain family. In another embodiment, an amino acid fragment may be replaced with a structurally similar fragment that differs in the order and composition of the side chain family members, e.g., conservative substitutions may be made in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
Amino acid residue families have been defined in the art with similar side chains including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
In some embodiments, the invention contemplates the production of functionally equivalent CAR polypeptide molecules, e.g., VH or VL of a humanized anti-P329G mutant scFv sequence comprised in a CAR can be modified to obtain a VH having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 12 and a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 13.
The transmembrane domain comprised in the CAR of the invention is an anchored transmembrane domain, which is a component of a polypeptide chain capable of integration in the cell membrane. The transmembrane domain may be fused to other extracellular and/or intracellular polypeptide domains, which will also be restricted to the cell membrane. In the Chimeric Antigen Receptor (CAR) polypeptides of the invention, the transmembrane domain confers membrane attachment to the CAR polypeptide of the invention. The CAR polypeptides of the invention comprise at least one transmembrane domain, which may be derived from natural sources or recombinant sources, comprising predominantly hydrophobic residues such as leucine and valine. Where the source is native, the domain may be derived from a membrane-binding protein or a transmembrane domain of a transmembrane protein such as CD28, CD8 (e.g., CD8 a, CD8 β). In one embodiment, the transmembrane domain comprises the amino acid sequence of SEQ ID NO. 15.
In some embodiments, the transmembrane domain in a CAR of the invention is linked to the extracellular region of the CAR (i.e., the humanized anti-P329G mutant scFv sequence) by a hinge/spacer region. For example, in one embodiment, the hinge may be a CD8 alpha hinge region, a CD28 hinge region. In some embodiments, the hinge region or spacer sequence comprises the amino acid sequence of SEQ ID NO. 18.
In addition, glycine-serine doublets also provide particularly suitable linkers as hinge/spacer regions. For example, in one embodiment, the linker comprises the amino acid sequence of GGGGS (SEQ ID NO: 14).
The cytoplasmic domain comprised in the CARs of the invention comprises an intracellular signaling domain. The intracellular signaling domain is capable of activating at least one effector function of an immune cell into which the CAR of the invention is introduced.
Examples of intracellular signaling domains for use in the CARs of the invention include cytoplasmic sequences of T Cell Receptors (TCRs) and co-receptors that act synergistically to initiate signal transduction upon binding of the extracellular domain to the P329G mutated antibody Fc domain, as well as any derivatives or variants of these sequences and any recombinant sequences having the same functional ability.
Given that the signal generated by the TCR alone is not yet sufficient to fully activate T cells, CARs of the invention also design a Costimulatory Signal Domain (CSD) capable of generating a costimulatory signal. Activation of T cells is mediated by two different classes of cytoplasmic signaling sequences: those sequences that initiate antigen-dependent primary activation by the TCR (primary intracellular signaling domains) and those sequences that function in an antigen-independent manner to provide a costimulatory signal (secondary cytoplasmic domains, e.g., costimulatory domains).
In one embodiment, a CAR of the invention comprises a primary intracellular signaling domain, e.g., a primary signaling domain of CD3δ, e.g., a CD3δ signaling domain as shown in SEQ ID NO: 17.
The intracellular signaling domain in the CARs of the invention also comprises a secondary signaling domain (i.e., a costimulatory signaling domain). A costimulatory signaling domain refers to the portion of the CAR that comprises the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules that are required by lymphocytes to respond effectively to antigens in addition to antigen receptors or their ligands. In some embodiments, costimulatory molecules include, but are not limited to, CD28, 4-1BB (CD 137), which cause costimulatory effects that enhance proliferation, effector function and survival of human CART cells in vitro and enhance antitumor activity of human T cells in vivo.
The intracellular signaling sequences in the CARs of the invention may be linked to each other in random order or in a specified order. Optionally, a short oligopeptide linker or polypeptide linker may form a bond between intracellular signaling sequences. In one embodiment, glycine-serine doublets may be used as suitable linkers. In one embodiment, a single amino acid, e.g., alanine, glycine, may be used as a suitable linker.
In one embodiment, the intracellular signaling domain of the CAR of the invention is designed to comprise the costimulatory signaling domain of 4-1BB and the stimulation signaling domain of cd3δ.
Nucleic acid molecules encoding the CARs of the invention, vectors and cells expressing the CARs of the invention
The invention provides nucleic acid molecules encoding the CAR constructs described herein. In one embodiment, the nucleic acid molecule is provided as a DNA construct.
The invention also provides vectors into which the CAR constructs of the invention are inserted. Expression of the natural or synthetic nucleic acid encoding the CAR is achieved by operably linking the nucleic acid encoding the CAR polypeptide to a promoter and incorporating the construct into an expression vector. Vectors may be suitable for replication and integration in eukaryotes. Common cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters for regulating the expression of the desired nucleic acid sequence.
Numerous virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene may be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus may then be isolated and delivered to cells of the subject in vivo or ex vivo. Numerous retroviral systems are known in the art. In some embodiments, lentiviral vectors are used.
Vectors derived from retroviruses (e.g., lentiviruses) are suitable tools for achieving long-term gene transfer, as they allow long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have the additional advantage over vectors derived from cancer-retroviruses (e.g., murine leukemia virus) in that they can transduce non-proliferative cells, such as hepatocytes. They also have the additional advantage of low immunogenicity. The retroviral vector may also be, for example, a gamma retroviral vector. The gamma retroviral vector may, for example, comprise a promoter, a packaging signal (ψ), a Primer Binding Site (PBS), one or more (e.g., two) Long Terminal Repeats (LTRs), and a transgene of interest, e.g., a gene encoding a CAR. The gamma retroviral vector may lack viral structural genes such as gag, pol and env.
An example of a promoter capable of expressing a CAR transgene in mammalian T cells is the EF1a promoter. The native EF1a promoter drives the expression of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNA to the ribosome. The EF1a promoter has been widely used in mammalian expression plasmids and has been shown to be effective in driving the expression of CARs from transgenes cloned into lentiviral vectors. See, e.g., milone et al, mol. Ther.17 (8): 1453-1464 (2009).
Another example of a promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. This promoter sequence is a constitutive strong promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. Other constitutive promoter sequences may be used including, but not limited to, monkey virus 40 (SV 40) early promoter, mouse Mammary Tumor Virus (MMTV), human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, moMuLV promoter, avian leukemia virus promoter, epstein barr virus immediate early promoter, rous sarcoma virus promoter, and human gene promoters such as, but not limited to, actin promoter, myosin promoter, elongation factor-1 alpha promoter, hemoglobin promoter, and creatine kinase promoter. In addition, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention.
In some embodiments, the invention provides methods of expressing the CAR constructs of the invention in mammalian immune effector cells (e.g., mammalian T cells or mammalian NK cells) and immune effector cells produced thereby.
A cell source (e.g., an immune effector cell, e.g., a T cell or NK cell) is obtained from a subject. The term "subject" is intended to include living organisms (e.g., mammals) that can elicit an immune response. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors.
T cells can be obtained from a blood component collected from a subject using any technique known to those skilled in the art (e.g., ficollTM isolation). In a preferred aspect, cells from circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes and platelets. In one embodiment, cells collected by apheresis may be washed to remove plasma fractions and to place the cells in a suitable buffer or medium for subsequent processing steps. In one aspect of the invention, the cells are washed with Phosphate Buffered Saline (PBS).
Specific T cell subsets, such as cd3+, cd28+, cd4+, cd8+, cd45ra+ and cd45ro+ T cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, the conjugate is provided by a bead conjugated to an anti-CD 3/anti-CD 28 (e.g.M-450 CD3/CD 28T) for a period of time sufficient to positively select the desired T cells, and isolating the T cells. In some embodiments, the period of time is between about 30 minutes and 36 hours or more. Longer incubation times can be used to isolate T cells in any situation where a small number of T cells are present, such as for isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals. In addition, the use of longer incubation times can increase the efficiency of cd8+ T cell capture. Thus, by simply shortening or lengthening this time, allowing T cells to bind to CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells, T cell subsets can be preferentially selected at the beginning of the culture or at other points in time during the culture process.
Enrichment of the T cell population can be accomplished by a negative selection process with a combination of antibodies directed against surface markers unique to the negatively selected cells. One method is to sort and/or select cells by means of a negative magnetic immunoadhesion method or flow cytometry using a monoclonal antibody mixture directed against cell surface markers present on the negatively selected cells.
In some embodiments, the immune effector cell may be an allogeneic immune effector cell, e.g., a T cell or NK cell. For example, the cells may be allogeneic T cells, e.g., allogeneic T cells lacking functional T Cell Receptors (TCRs) and/or expression of Human Leukocyte Antigens (HLA) (e.g., HLA class I and/or HLA class II).
A T cell lacking a functional TCR may, for example, be engineered so that it does not express any functional TCR on its surface; engineered so that it does not express one or more subunits that make up a functional TCR (e.g., engineered so that it does not express or exhibit reduced expression of tcra, tcrp, tcrγ, tcrδ, tcrε, and/or tcrδ); or engineered so that it produces very few functional TCRs on its surface.
The T cell described herein may, for example, be engineered such that it does not express a functional HLA on its surface. For example, T cells described herein can be engineered such that cell surface expression of HLA (e.g., HLA class I and/or HLA class II) is down-regulated. In some aspects, down-regulation of HLA can be achieved by reducing or eliminating beta-2 microglobulin (B2M) expression.
In some embodiments, T cells may lack a functional TCR and a functional HLA, e.g., HLA class I and/or HLA class II.
In one embodiment, the nucleic acid-transduced cells encoding the CARs of the invention are propagated, e.g., the cells are propagated in culture for 2 hours to about 14 days.
The immune effector cells expressing the CAR obtained after in vitro proliferation can be tested for effector function as described in the examples.
In a specific embodiment, the first component suitable for use in the pharmaceutical combination of the invention is a CAR-T cell comprising a CAR of the invention.
Antibodies that specifically bind BCMA molecules and antibodies comprising mutant Fc domains
The present invention provides an antibody that binds BCMA with high target specificity and high affinity comprising a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region comprises CDR H1 according to amino acid sequence SSSYYWT of Kabat numbering (SEQ ID NO: 25), or a variant of said CDR H1 that does not vary by more than 2 amino acids or by more than 1 amino acid; CDR H2 represented by amino acid sequence SISIAGSTYYNPSLKS (SEQ ID NO: 26), or a variant of said CDR H2 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid; and amino acid sequence DRGDQILDV (SEQ ID NO: 27), or a variant of said CDR H3 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid; the light chain variable region comprises a CDR L1 as set forth in amino acid sequence RASQSISRYLN (SEQ ID NO: 28) according to Kabat numbering, or a variant of said CDR L1 with NO more than 2 amino acid changes or NO more than 1 amino acid change; CDR L2 represented by amino acid sequence AASSLQS (SEQ ID NO: 29), or a variant of said CDR L2 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid; and amino acid sequence QQKYFDIT (SEQ ID NO: 30), or a variant of said CDR L3 that does not vary by more than 2 amino acids or does not vary by more than 1 amino acid;
wherein the amino acid change is an addition, deletion or conservative amino acid substitution of an amino acid.
In some embodiments, antibodies that bind BCMA molecules of the invention bind mammalian BCMA, e.g., human, cynomolgus monkey, mouse, rat, and rabbit BCMA.
In some embodiments, antibodies of the invention that bind BCMA molecules have one or more of the following properties:
(1) Specifically binds BCMA;
(2) Binds human BCMA and cross-reacts with cynomolgus monkey, mouse, rat and rabbit BCMA;
(3) In the case of non-inclusion of a mutant Fc domain, e.g., having a parent antibody Fc domain that is not mutated, BCMA-positive cancer cells can be killed by antibody dependent cytotoxicity and/or Antibody Dependent Cellular Phagocytosis (ADCP).
In some embodiments, antibodies that bind to BCMA molecules of the invention comprise a heavy chain variable region comprising the sequence of SEQ ID NO:2 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, wherein the amino acid change in the sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity is preferably an amino acid substitution, more preferably an amino acid conservative substitution, preferably the amino acid change does not occur in the CDR region.
In some embodiments, antibodies that bind to BCMA molecules of the invention comprise a light chain variable region comprising the sequence of SEQ ID NO:3 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, wherein the amino acid change in the sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity is preferably an amino acid substitution, more preferably an amino acid conservative substitution, preferably the amino acid change does not occur in the CDR region.
In some embodiments, an antibody that binds a BCMA molecule of the invention comprises a heavy chain variable region and a light chain variable region, wherein:
the heavy chain variable region comprises the sequence of SEQ ID No. 2 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain variable region comprises the sequence of SEQ ID No. 3 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
Wherein the amino acid change in the sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical is preferably an amino acid substitution, more preferably an amino acid conservative substitution, preferably the amino acid change does not occur in a CDR region.
In some embodiments, an antibody that binds a BCMA molecule of the invention comprises a heavy chain variable region comprising or consisting of the sequence of SEQ ID No. 2 and a light chain variable region comprising or consisting of the sequence of SEQ ID No. 3.
In some embodiments, the antibodies that bind BCMA molecules of the invention are IgG1, igG2, igG3, or IgG4 antibodies; preferably, it is an IgG1 or IgG4 antibody; more preferably, it is an IgG1 antibody, e.g., a human IgG1 antibody.
In some embodiments, an antibody that binds a BCMA molecule provided herein comprises a mutant Fc domain wherein the amino acid at position P329 according to EU numbering is mutated to glycine (G) and the fcγ receptor binding of the mutant Fc domain is reduced compared to the fcγ receptor binding of the unmutated parent antibody Fc domain; for example, the mutant Fc domain is a mutant Fc domain of an IgG1, igG2, igG3, or IgG4 antibody, preferably the mutant Fc domain is a mutant Fc domain of an IgG1 or IgG4 antibody; more preferably, the mutant Fc domain is a mutant Fc domain of an IgG1 antibody, e.g., the mutant Fc domain is a mutant Fc domain of a human IgG1 antibody. In some embodiments, the mutant Fc domain comprises the amino acid sequence of SEQ ID No. 10, or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to said amino acid sequence, and comprises a P329G mutation.
In some embodiments, antibodies that bind BCMA molecules comprising a P329G mutant Fc domain are not capable of exerting antibody dependent cellular cytotoxicity by binding to fcγ receptor, nor are they capable of exerting Antibody Dependent Cellular Phagocytosis (ADCP).
In some embodiments, antibodies that bind to BCMA molecules of the invention comprise a heavy chain wherein the heavy chain variable region comprises the sequence of SEQ ID NO:34 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto and comprises P329G (EU numbering), wherein the amino acid change in the sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity is preferably an amino acid substitution, more preferably an amino acid conservative substitution, preferably the amino acid change does not occur in the CDR region or does not occur in the variable region.
In some embodiments, antibodies that bind to BCMA molecules of the invention comprise a light chain, wherein the light chain variable region comprises the sequence of SEQ ID NO:35 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, wherein an amino acid change in the sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity is preferably an amino acid substitution, more preferably an amino acid conservative substitution, preferably the amino acid change does not occur in the CDR region or does not occur in the variable region.
In some embodiments, an antibody that binds a BCMA molecule of the invention comprises a heavy chain and a light chain, wherein:
The heavy chain comprises the sequence of SEQ ID No. 34 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain comprises the sequence of SEQ ID No. 35 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;
Wherein the amino acid change in the sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity is preferably an amino acid substitution, more preferably an amino acid conservative substitution, preferably the amino acid change does not occur in the CDR region or variable region.
In some embodiments, antibodies that bind BCMA molecules of the invention comprise a heavy chain comprising or consisting of the sequence of SEQ ID No. 34 and a light chain comprising or consisting of the sequence of SEQ ID No. 35.
In some embodiments, the invention provides nucleic acids encoding any of the above antibodies or fragments thereof that bind to BCMA molecules or any one of the chains thereof. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising the nucleic acid or the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell (e.g., CHO cell or 293 cell) or other cell suitable for the production of antibodies or antigen binding fragments thereof. In another embodiment, the host cell is prokaryotic.
For example, the nucleic acids of the invention comprise nucleic acids encoding antibodies of the invention that bind BCMA molecules. In some embodiments, one or more vectors comprising the nucleic acid are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or Yeast Artificial Chromosomes (YACs). In one embodiment, the vector is a pcDNA3.4 expression vector.
Once the expression vector or DNA sequence has been prepared for expression, the expression vector may be transfected or introduced into a suitable host cell. Various techniques may be used to achieve this, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, the cells are incubated in medium and screened for appropriate activity. Methods and conditions for culturing the resulting transfected cells and for recovering the resulting antibody molecules are known to those skilled in the art and may be varied or optimized depending on the particular expression vector and mammalian host cell used, based on the present specification and methods known in the art.
Alternatively, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. The marker may, for example, provide prototrophy, biocidal resistance (e.g., antibiotics) or heavy metal (e.g., copper) resistance to an auxotrophic host, and the like. The selectable marker gene may be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements may include splicing signals, transcriptional promoters, enhancers, and termination signals.
In one embodiment, a host cell comprising a polynucleotide of the invention is provided. In some embodiments, host cells comprising the expression vectors of the invention are provided. In some embodiments, the host cell is selected from a yeast cell, a mammalian cell, or other cell suitable for the production of antibodies. Suitable host cells include prokaryotic microorganisms, such as E.coli. The host cell may also be a eukaryotic microorganism such as a filamentous fungus or yeast, or various eukaryotic cells, e.g., insect cells, etc. Vertebrate cells can also be used as hosts. For example, mammalian cell lines engineered to be suitable for suspension growth may be used. Examples of useful mammalian host cell lines include the SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney (HEK 293 or 293F cells), 293 cells, baby Hamster Kidney (BHK), monkey kidney (CV 1), african green monkey kidney (VERO-76), human cervical cancer (HELA), canine kidney (MDCK), buffalo rat liver (BRL 3A), human lung (W138), human liver (HepG 2), chinese Hamster Ovary (CHO) cells, CHO-S cells, NSO cells, myeloma cell lines such as Y0, NS0, P3X63 and Sp2/0, etc. For a review of mammalian host cell lines suitable for the production of proteins see, e.g., yazaki and Wu, methods in Molecular Biology, vol.248 (b.K.C.Lo et al, humana Press, totowa, NJ), pages 255-268 (2003). In a preferred embodiment, the host cell is a CHO cell or HEK293 cell.
In one embodiment, the invention provides a method of making an antibody that binds BCMA molecules (including a P329G mutant antibody), wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody that binds BCMA molecules (including a P329G mutant antibody) or an expression vector comprising the nucleic acid under conditions suitable for expressing a nucleic acid encoding the antibody that binds BCMA molecules (including a P329G mutant antibody), and optionally isolating the antibody that binds BCMA molecules (including a P329G mutant antibody). In a certain embodiment, the method further comprises recovering antibodies (including P329G mutant antibodies) that bind BCMA molecules from the host cell (or host cell culture medium).
Antibodies (including P329G mutant antibodies) that bind BCMA molecules of the present invention prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and these will be apparent to those skilled in the art. The purity of antibodies (including P329G mutant antibodies) that bind BCMA molecules of the present invention can be determined by any of a variety of well-known analytical methods including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.
Antibodies (including P329G mutant antibodies) that bind BCMA molecules provided herein can be identified, screened, or characterized for physical/chemical properties and/or biological activity by a variety of assays known in the art. In one aspect, antibodies of the invention that bind BCMA molecules (including P329G mutant antibodies) are tested for antigen binding activity, for example, by known methods such as FACS, ELISA, or Western blot. Binding to BCMA can be determined using methods known in the art, exemplary methods are disclosed herein. In some embodiments, binding of antibodies (including P329G mutant antibodies) that bind to BCMA molecules of the invention to cell surface BCMA (e.g., human BCMA) is determined using FACS.
The invention also provides assays for identifying antibodies (including P329G mutant antibodies) that bind BCMA molecules having biological activity. Biological activities may include, for example, ADCC, CDC, etc.
Cells for use in any of the in vitro assays described above include cells that naturally express BCMA or are engineered to express BCMA. The cell line engineered to express BCMA is a cell line that expresses BCMA after transfection of DNA encoding BCMA into cells that normally does not express BCMA.
V. pharmaceutical composition of the invention
For optimizing the safety and efficacy of CAR therapies, the molecular switch-regulated chimeric antigen receptor of the invention is a regulated CAR that can control CAR activity. The present invention uses a mutant antibody in which Pro329Gly (proline at position 329 of the Fc fragment of the antibody is mutated to glycine according to EU numbering, abbreviated as P329G) as a safety switch in the treatment of the CAR of the present invention. In the absence of the P329G mutant antibody, the CAR activity of the invention is turned off; in the presence of the P329G mutant antibody, the CAR activity of the invention is turned on; thus, the turning on and off of CAR molecule activity of the present invention is regulated by P329G mutant antibodies.
In some embodiments, the invention provides a pharmaceutical combination comprising (i) an immune effector cell (e.g., T cell, NK cell) expressing a molecular switch-regulated CAR polypeptide of the invention; and (ii) a P329G mutant antibody that specifically binds to a BCMA molecule. For example, the immune effector cell is a T cell expressing a molecular switch-regulated CAR polypeptide of the invention prepared from autologous T cells or allogeneic T cells, e.g., the immune effector cell is a T cell expressing a molecular switch-regulated CAR polypeptide of the invention prepared from T cells isolated from human PBMCs. In some embodiments, the P329G mutant antibody is ADI-38497 PG Ab.
In some embodiments, the invention provides a pharmaceutical combination comprising (i) a nucleic acid molecule encoding a molecular switch-regulated CAR polypeptide of the invention or a vector comprising the nucleic acid component; and (ii) a P329G mutant antibody that specifically binds to a BCMA molecule.
In some embodiments, the pharmaceutical combination of the invention optionally further comprises pharmaceutically acceptable excipients of a suitable formulation. For example, (ii) of the pharmaceutical combination may be formulated according to conventional methods (e.g. Remington's Pharmaceutical Science, latest edition, mark Publishing Company, easton, u.s.a.).
In some embodiments, the pharmaceutical combinations of the invention are used to treat a disease associated with BCMA, e.g., BCMA expressing or overexpressing cancer, e.g., multiple myeloma, wherein the multiple myeloma is refractory multiple myeloma or relapsed multiple myeloma, e.g., the multiple myeloma is relapsed/refractory multiple myeloma (relapsed/refractory multiple myeloma, RRMM).
Use of the pharmaceutical combinations of the invention and methods of treatment using the pharmaceutical combinations of the invention
The present invention provides the aforementioned pharmaceutical combination of the invention for use in the treatment of a disease associated with BCMA, such as a BCMA expressing or overexpressing cancer, such as relapsed/refractory multiple myeloma (relapsed/refractory multiple myeloma, RRMM) in a subject.
In one embodiment, the pharmaceutical combination of the invention is used to treat a cancer that expresses or overexpresses BCMA in a subject and is capable of reducing the severity of at least one symptom or indication of cancer or inhibiting cancer cell growth, said cancer being multiple myeloma, e.g., refractory multiple myeloma or relapsed multiple myeloma, such as relapsed/refractory multiple myeloma.
The invention provides methods of treating a disease associated with BCMA (e.g., BCMA expressing or overexpressing cancer, such as multiple myeloma, e.g., refractory multiple myeloma or relapsed multiple myeloma, such as relapsed/refractory multiple myeloma) in a subject, comprising administering to an individual in need thereof a therapeutically effective amount of a pharmaceutical combination of the invention.
The present invention provides the use of a pharmaceutical combination of the foregoing invention in the manufacture of a medicament for the treatment of a disease associated with BCMA (e.g., BCMA expressing or overexpressing cancer, such as multiple myeloma, e.g., refractory multiple myeloma or relapsed multiple myeloma, e.g., relapsed/refractory multiple myeloma).
The pharmaceutical combinations of the invention may also be administered to an individual who has been treated for cancer with at least three previous therapies but subsequently relapsed or metastasized, e.g., the cancer is multiple myeloma, e.g., refractory multiple myeloma or relapsed multiple myeloma, such as relapsed/refractory multiple myeloma (relapsed/refractory multiple myeloma, RRMM). In some embodiments, prior therapies include proteasome inhibitor and immunomodulator treatments.
In some embodiments, the multiple myeloma is a refractory multiple myeloma (refractory multiple myeloma). In some embodiments, wherein the multiple myeloma is relapsed multiple myeloma (relapsed multiple myeloma). In some embodiments, the multiple myeloma is refractory and relapsed multiple myeloma relapse/refractory multiple myeloma (relapsed/refractory multiple myeloma, RRMM). In some embodiments, the subject has received at least three prior therapies for treating multiple myeloma. In some embodiments, prior therapies include proteasome inhibitor and immunomodulator treatments.
In some embodiments, an immune effector cell (e.g., T cell, NK cell) expressing a molecular switch-regulated CAR polypeptide of the invention and (ii) a P329G mutant antibody that specifically binds to a BCMA molecule in a pharmaceutical combination of the invention are for parenteral, transdermal, intracavitary, intraarterial, intravenous, intrathecal administration, or direct injection into a tissue or tumor. In some embodiments, the (ii) P329G mutant antibody that specifically binds to a BCMA molecule in a pharmaceutical combination of the invention is administered prior to (i) an immune effector cell (e.g., T cell, NK cell) that expresses a molecular switch-regulated CAR polypeptide of the invention.
In some embodiments, the (i) immune effector cells expressing the molecular switch-regulated CAR polypeptides of the invention in the pharmaceutical combination of the invention are T cells prepared from autologous T cells or allogeneic T cells that express the CAR polypeptides of the invention; the (ii) P329G mutant antibody that specifically binds to a BCMA molecule in the pharmaceutical combination of the present invention is any antibody that specifically binds to a BCMA molecule comprising a P329G mutation. Preferably, the P329G mutant antibody is ADI-38497 PG Ab.
In some embodiments, administration of the pharmaceutical combination of the invention to an individual suffering from cancer results in complete disappearance of the tumor. In some embodiments, administration of a pharmaceutical combination of the invention to an individual having cancer results in a reduction in tumor cell or tumor size of at least 85% or more. The reduction of tumors may be measured by any method known in the art, such as X-ray, positron Emission Tomography (PET), computed Tomography (CT), magnetic Resonance Imaging (MRI), cytology, histology, or molecular genetic analysis.
In some embodiments, the pharmaceutical combinations of the invention can reduce the "On-target/off tumor" toxicity of the presence of CAR-T cells.
VII the therapy of the invention
In some embodiments, the invention relates to a method of treating multiple myeloma in a subject, comprising administering to the subject a pharmaceutical combination comprising
(I) The first component, for example, is a T cell expressing a molecular switch-regulated CAR polypeptide, i.e., a CAR-T cell.
(Ii) A second component, for example, which is an antibody or antigen binding fragment that specifically binds to a BCMA molecule comprising a P329G mutation, wherein the P329G mutant antibody comprises a mutant Fc domain wherein the amino acid at position P329 according to EU numbering is mutated to glycine (G).
In some embodiments, the method comprises administering the first component at a dose of about 0.5X106 cells/kg body weight to about 5X 106 cells/kg body weight, e.g., about 0.75X106 cells/kg body weight, 1X 106 cells/kg body weight, 1.5X106 cells/kg body weight, 2X 106 cells/kg body weight, 2.5X106 cells/kg body weight, 3X 106 cells/kg body weight, 3.5X106 cells/kg body weight, 4X 106 cells/kg body weight, 4.5X106 cells/kg body weight, 5X 106 cells/kg body weight to the subject. In some embodiments, the administration is a single administration or multiple administrations, preferably a single administration. In some embodiments, the administration is intravenous administration.
In some embodiments, the method comprises administering the second component in a dosage form of a dosage unit of about 0.1-3mg/kg, preferably about 0.1mg/kg, 0.2mg/kg, 0.3mg/kg, 0.4mg/kg, 0.5mg/kg, 1mg/kg, 1.5mg/kg, 2mg/kg, 2.5mg/kg, 3 mg/kg. In some embodiments, the administration is parenteral, more preferably intravenous administration.
In some embodiments, the first component and the second component are administered in the following regimen:
the second component is administered on the first day, with the first component administered about 1-5 hours apart (e.g., about 1,2,3, 4, or 5 apart); or the second component is administered on the first day, the first component is administered on the second day, about 24 hours apart;
taking the first day as the starting date, the second component is administered every 1 week (QW), 2 weeks (Q2W), or 3 weeks (Q3W) in the first period;
second to nth cycles: the second component was administered every three weeks (Q3W) in each cycle.
In some embodiments, each dosing cycle is 21 days or 28 days.
In some embodiments, at least 2-3 cycles of administration. In some embodiments, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cycles or more are administered.
In some embodiments, the subject is administered a Lymphoscavenging (LD) regimen. In some embodiments, a Lymphotropic (LD) regimen is administered to the subject prior to administration of the second component. In some embodiments, the lymphocidal regimen is administered prior to administration of the second component, e.g., beginning administration 1, 2, 3, 4, 5, or 6 days prior to administration. In some embodiments, the duration of the LD is about 1 to 5 days, for example about 3 days. In some embodiments, the time window between the end of LD and the beginning of administration of the second component, e.g., BCMA antibody, is 2 days or 3 days or 4 days. In some embodiments, LD begins about 3 to 5 before a dose of a second component, such as a BCMA antibody, is administered.
In some embodiments, LD is started about 3,4, or 5 days prior to administration of the second component, e.g., anti-BCMA antibody dose. In some embodiments, a dose of a second component, e.g., an anti-BCMA antibody, is administered about 1,2, or 3 days after LD is complete.
In some embodiments, the LD comprises fludarabine and cyclophosphamide.
In some embodiments, the first component, e.g., CAR-T cells, are prepared within about 20-40 days prior to administration of the second component of the invention. In some embodiments, the first component, e.g., CAR-T cells, are prepared in about 25-35 days. In some embodiments, the first component comprises T cells. In some embodiments, the T cells are autologous T cells, e.g., T cells obtained from a blood component collected from a subject.
In some embodiments, the invention relates to a pharmaceutical combination for use in the methods of treatment of the invention.
In some embodiments, the subject has a low probability of developing CRS or ICANS, e.g., less than 50%, e.g., less than an existing BCMA-CAR-T product, following administration of the therapy of the present invention.
In some embodiments, the therapies of the invention have good safety and/or tolerability.
In some embodiments, the second component (e.g., an anti-BCMA antibody) is capable of modulating the activity of the first component, particularly the cellular activity of the CAR-T cells.
In some embodiments, the therapies of the invention are capable of partially alleviating a disease in a subject, and clinical studies have shown that a panel of 11 patients are pooled, with 5 patients achieving PR and above remission.
VIII the medicine box of the invention
The present invention provides a kit comprising a pharmaceutical combination according to the invention, preferably in the form of a pharmaceutical dosage unit. The dosage units may thus be provided according to the dosing regimen or the interval between drug administrations.
In one embodiment, the kit of parts of the invention comprises, in the same package:
(i) An immune effector cell (e.g., T cell, NK cell) selected from the group consisting of a molecule switch-regulated CAR polypeptide of the invention, a nucleic acid molecule encoding a molecule switch-regulated CAR polypeptide of the invention, a vector comprising the nucleic acid, and any combination thereof;
(ii) P329G mutant antibodies that specifically bind BCMA molecules.
The various embodiments/technical solutions described herein and features in the various embodiments/technical solutions should be understood to be arbitrarily combined with each other, and the various solutions obtained by these combinations are included in the scope of the present invention as if the combinations were specifically and individually listed herein unless the context clearly indicates otherwise.
The following examples are described to aid in the understanding of the present invention. The examples are not intended to, and should not be construed in any way as, limiting the scope of the invention.
Examples
Example 1, CAR Gene Synthesis, construction of viral expression vector, preparation of P329G CAR-T cells and detection of CAR expression
(1-1) CAR Gene Synthesis and construction of viral expression vectors
P329G CAR molecules (SEQ ID NO: 1), also known as HuR968B CAR, were constructed from fusion of the Signal Peptide (SP) shown in SEQ ID NO:11, a specific single chain antibody fragment (VH-linker-VL having the VH shown in SEQ ID NO:12, the linker sequence shown in SEQ ID NO:33, the VL shown in SEQ ID NO: 13), the G4S hinge region shown in SEQ ID NO:14, the CD8 transmembrane domain (CD 8 TM) shown in SEQ ID NO:15, the 41BB co-stimulatory domain (41 BB-CSD) shown in SEQ ID NO:16, and the CD3 delta molecule intracellular activation domain (CD 3 delta SSD) shown in SEQ ID NO: 17.
In addition, a direct BCMA-targeted Blue21 CAR (SEQ ID NO: 8) was constructed for use as a control. Blue21 CAR contains from N-terminus to C-terminus the signal peptide shown in SEQ ID NO. 11, an anti-BCMA single chain antibody (from 11D53 clone), the hinge region of the CD 8. Alpha. Molecule shown in SEQ ID NO. 18, the CD8 transmembrane domain shown in SEQ ID NO. 15, the 4-1BB co-stimulatory domain shown in SEQ ID NO. 16 and the CD3δ chain intracellular activation domain shown in SEQ ID NO. 17.
The above DNA fragment encoding the CAR polypeptide was inserted into the downstream of EF 1a promoter of pRK lentiviral expression vector (modified by prrlsin. Cppt. Pgk-gfp. Wpre vector (Addgene, 12252, purchased from bio-wind)) by replacing promoter and resistance gene, and EGFR sequence in the vector was replaced, to obtain CAR expression plasmid pRK-HuR968B, pRK-Blue21.
(1-2) Preparation of lentiviral concentrate
The CAR expression plasmid prepared in example 1-1 was transfected with structural plasmid pMDLg/pRRE (Addgene, 12251, purchased from biofuels), regulatory plasmid pRSV-rev (Addgene, 12253, purchased from biofuels) and envelope plasmid pMD2G (Addgene, 12259, purchased from biofuels) at a mass ratio of 3:3:2:2 by PEI transfection method, and after 16 hours of transfection, replaced with fresh DEME medium containing 2% Fetal Bovine Serum (FBS), after further culturing for 48 hours, cell supernatants were collected, centrifuged to remove cell debris, PEG8000 4 ℃ was added for incubation for 16-64 hours for lentiviral concentration, centrifuged again to remove the supernatant, and the lentiviral pellet was resuspended with T cell medium to obtain lentiviral concentrate, which was frozen at-80 ℃ after sub-packaging.
(1-3) P329G CAR-T cell preparation and CAR expression detection:
recombinant human interleukin-2 (national standard S20040020) for injection was added to TexMACS GMP Medium (Miltenyi Biotec, 170-076-309) to prepare a T cell culture medium with an IL-2 concentration of 200 IU/ml.
A plurality of donor PBMC cells were obtained from ORiCELLS, the specific information being shown in table 1 below:
TABLE 1 relevant Source information of donor PBMC cells
On day 0, each donor PBMC after resuscitation was sorted using Pan T Cell Isolation Kit (human) (Miltenyi, 130-096-535) to obtain T cells, which were resuspended to a certain density using T cell medium and activated by addition of TransAct (Miltenyi, 130-111-160).
Continuing culturing by removing a certain amount of T cells on day 1 without adding lentiviral concentrate, wherein the part of the cells are untransduced cells (UNT, un-transduced T cells), adding the lentiviral concentrate obtained from example 1-2 (the lentivirus is a lentivirus encoding P329G CAR (SEQ ID NO: 1) or a control traditional CAR (SEQ ID NO: 8) according to MOI=1-5 to the rest of the T cells, and blowing the T cells uniformly; the virus supernatant was removed by centrifugation on day 2 and the cells were resuspended in fresh T cell medium. All cells were transferred to G-Rex (WILSONWOLF, cat# 80040S) on day 3, and appropriate amount of fresh T cell medium was added, placed in a CO2 incubator at 37 ℃ for stationary culture; every 2-3 days, the cells are replaced by fresh culture medium with half of the culture medium or are directly supplemented with IL-2, wherein the IL-2 is added to the cell culture medium, and the concentration of the IL-2 in the cell culture medium is 200IU/ml. When the number of cells is expanded to about 20-80 times, cell harvesting is performed after the demand is satisfied (typically up to 2-8 x108 cells). After centrifugation of the medium, CAR-T cells were subjected to the following procedureCS10 (Stemcell, 07930) was resuspended and then sub-packaged, and the temperature was programmed to-80 ℃ for cryopreservation.
Washing a proper amount of CAR-T cells once by using FACS buffer (PBS+2% FBS), adding the FACS buffer containing LIVE/DEAD Fixable DEAD CELL STAIN after re-suspending, staining at room temperature for 10-15min, washing twice, adding PerCP-Cy5.5-CD3, BUV805-CD, biotin-F (ab ')2 Fragment goat anti-human IgG (Jackson ImmunoResearch,109-066-006; PG CAR detection) or Biotin-F (ab')2 Fragment goat anti-mouse IgG (Jackson ImmunoResearch,115-066-006; blue 21CAR detection) antibody combination, staining at 4 ℃ for 30-45 min, washing twice, and then adding APC-strepitavidin for staining at 4 ℃ for 30-45 min; after washing twice, the cells were resuspended in FACS buffer and examined using a flow cytometer.
FIG. 1A shows the expression of CARs in CD3+ cells, CD4+、CD8+ T cell subsets after T cell transduction with 2 CARs constructed in example 1-1, respectively, and shows that the positive rate of CAR expression in these transduced T cells is approximately 18% to 29%.
Example 2 detection of antigen binding Activity of BCMA-specific P329G mutant antibodies
(2-1) Synthesis of BCMA-specific antibodies
The heavy and light chain variable region sequences of the BCMA parent antibody ADI-34861 (VH shown in SEQ ID NO:31, VL sequence shown in SEQ ID NO:32, respectively) were obtained from International application No. PCT/CN2019/074419 (BCMA antibody-related patent), and CDR region mutations were performed on the basis of the parent antibody ADI-34861 to obtain the heavy and light chain variable region sequences of ADI-38497 (SEQ ID NO:2, SEQ ID NO: 3). The mutated antibodies showed a significant improvement in affinity compared to the corresponding parent antibodies, and the specific experimental data are shown in table 2 below.
Table 2 parent and mutant antibodies binding affinities to BCMA
The GSK company BCMA antibody clone J6M0 light and heavy chain variable region sequence was obtained from US9273141B2 patent as a control antibody (GSK IgG).
The GSK IgG and ADI-38497 antibody light and heavy chain variable region sequences are synthesized by adopting total genes, and are loaded on pcDNA3.4 expression vector (purchased from Shanghai primary English) containing the WT human IgG1 heavy chain constant region (SEQ ID NO: 4) or the human IgG1 heavy chain constant region (SEQ ID NO: 5) containing P329G point mutation and kappa light chain constant region (SEQ ID NO: 6). Light and heavy chain expression vectors are co-transfected into HEK293 cells through PEI according to a molar ratio of 2:3, and culture medium supernatants are collected after 5-7 days of culture. The supernatant medium containing the antibodies was purified in one step by Protein a column, after which it was dialyzed against PBS. The concentration was measured by reading the absorbance at 280nm using a NanoDrop instrument and the purity of the samples was measured by SDS-PAGE and SEC-HPLC methods. Obtaining GSK WT antibody and GSK PG antibody; ADI-38497 WT antibody and ADI-38497 PG antibody. Antibodies having the sequences of the heavy chain variable region (SEQ ID NO: 2) and the light chain variable region (SEQ ID NO: 3) of the BCMA antibody clone ADI-38497 are also referred to herein as ADI-38497 antibodies, including ADI-38497 PG antibodies (comprising the P329G mutation) and ADI-38497 WT antibodies.
(2-2) Affinity detection of antibodies
The affinity of ADI-38497 PG antibodies to BCMA of different species was determined by Biacore T200 and figure 2A shows a schematic diagram of the method for determining antibody affinity using Surface Plasmon Resonance (SPR).
The specific method comprises the following steps: after coupling anti-human Fc IgG (Ab 97221, abcam) to the surface of CM5 chips (2914953, cytova), the ADI-38497 PG antibodies were captured on the chip surface and affinity and kinetic constants were obtained by detecting binding and dissociation between the chip surface antibodies and BCMA antigen in the mobile phase. The assay procedure used 10 XHBS-EP+ (BR-1006-69, cytiva) after 10-fold dilution as the assay buffer. Each cycle in affinity detection involves capture of ADI-38497 PG antibody, binding to one concentration of antigen, and chip regeneration. The antigen after gradient dilution (antigen concentration gradient 1.25-40nM, 2-fold dilution) was flowed through the chip surface from low concentration to high concentration in order of 30. Mu.l/min, and the binding time was 180s, and the appropriate dissociation time (900 s or 600s or 60 s) was set. Finally, the chip was regenerated using 10mM glycine-HCl, pH 1.5 (BR-1003-54, cytiva).
The data results were analyzed by Biacore T200 analysis software (version number 3.1) using the 1:1 binding model.
FIG. 2B shows a representative affinity profile of ADI-38497 PG antibodies to recombinant human, cynomolgus monkey, mouse, rat and rabbit BCMA proteins using SPR. The results show that ADI-38497 PG antibody can be combined with BCMA proteins from different species, wherein the sequence of the combining activity is that human BCMA > monkey BCMA > mouse BCMA > rat BCMA > rabbit BCMA.
Table 3 ADI-38497 PG antibodies binding affinities to BCMA proteins from different species
(2-3) Detection of P329G BCMA antibodies and detection of different genus-derived BCMA antigen binding Activity
First, CHO GS cells expressing BCMA antigens from different species were prepared. Specifically, human, mouse, cynomolgus BCMA genes were synthesized and cloned into lentiviral vectors, then lentiviruses containing BCMA genes of different species were packaged, CHO GS cells were infected with the lentiviruses, and then flow cytometry sorting was performed to obtain CHO GS cell lines expressing BCMA antigens of different species, i.e., hbma-CHO GS, mBCMA-CHO GS and cynoBCMA-CHO GS cells.
Then, ADI-38497 PG antibodies and GSK-derived BCMA antibodies (i.e., GSK PG IgG was used as a Benchmark) were formulated as 10-fold gradient dilutions of different concentration antibody solutions with FACS buffer, incubated with 1E5 prepared CHO GS cells expressing different species-derived BCMA antigens at 4℃for 30min, washed with FACS buffer, and incubated with Fcγ fragment-specific APC-goat anti-human IgG (Jackson ImmunoResearch, 109-136-098) at 4℃for 30 min. Cell-bound P329G antibodies were detected by flow cytometry, APC channel MFI was analyzed, plotted on the X-axis with antibody concentration and on the Y-axis and the EC50 of binding was calculated.
FIG. 2C shows the binding capacity of different concentrations of P329G BCMA antibodies to CHO-GS cells stably expressing human, cynomolgus and mouse BCMA. As can be seen from fig. 2C, ADI-38497 PG IgG antibody was able to bind to different species of BCMA expressed on the cell surface, whereas GSK-derived BCMA antibody (Benchmark) had higher species BCMA specificity, which did not recognize mouse BCMA, consistent with the SPR detection results.
TABLE 4 EC50 values for binding of P329G BCMA antibodies to CHO-GS cells expressing different species of BCMA
(2-4) Detection of the binding Activity of P329G BCMA antibodies to tumor cell surface BCMA antigen
An appropriate amount of tumor cells in the logarithmic growth phase were washed 2 times with FACS buffer, ADI-38497 PG antibody and GSK PGIgG as a Benchmark were added, cells as a staining control were stained with isotype hIgG1 antibody at 4℃for 30 minutes, washed twice, goat anti-human IgG antibody of APC-F (ab')2 fragment was added, stained at 4℃for 30 minutes, and cells were resuspended with FACS buffer after washing twice, and detected with a flow cytometer.
FIG. 2D shows the binding activity of different concentrations of P329G BCMA antibodies to BCMA expressing positive multiple myeloma cell lines MM.1s, RPMI8226, U266, H929, L363 and AMO1 (MM.1 s from Nanj, bai Biotechnology Co., ltd., CBP60239; RPMI8226 from Nanj, bai Biotechnology Co., ltd., CBP60244; U266 from Tokunux Siro life technologies Co., CL-0510; H929 from Nanj Bai Biotechnology Co., ltd., CBP60243; L363, bai Biotechnology Co., ltd., CBP6024; AMO1 from Nanj Biotechnology Co., ltd., CBP 60242), ADI-38497 PG antibodies being able to bind to BCMA expressing positive tumor cells and exhibiting concentration dependence. Of the positive tumor cells expressing BCMA, mm.1s cells had the highest level of BCMA expression, RPMI8226, U266 and H929 cells expressed BCMA at medium level, and L363, AMO1 cells expressed BCMA at low level.
TABLE 5 EC50 values for binding of P329G BCMA antibodies to BCMA-expressing positive tumor cells
Example 3 detection of binding Activity of BCMA-specific P329G mutant antibodies to the extracellular Domain of P329G CARs
(3-1) Affinity detection of P329G mutant antibodies binding to the P329G CAR extracellular domain
The binding affinity of ADI-38497 molecules (P329G mutant antibodies or wild type antibodies) to anti-P329G mutation specific single chain antibodies-rabbit Fc fusion proteins (also referred to as "anti-PG scFv fusion proteins" or simply "anti-PG scFv") was determined by Biacore T200. FIG. 3A shows a schematic assay for determining affinity of a specific single chain antibody-rabbit Fc fusion protein against the P329G mutation to an ADI-38497P 329G mutant antibody using Surface Plasmon Resonance (SPR).
The specific method comprises the following steps: after coupling the synthesized anti-PG scFv fusion protein (SEQ ID NO: 7) to the surface of a C1 chip (BR 100535, cytiva), affinity and kinetic constants were obtained by detecting the binding and dissociation of the antibody on the chip surface to the ADI-38497 molecule in the mobile phase. The assay procedure used 10 XHBS-EP+ (BR-1006-69, cytiva) after 10-fold dilution as the assay buffer. Each cycle of affinity detection involves binding of one concentration of antibody and chip regeneration. ADI-38497 molecules (concentration gradient 3.125nM-100nM, 2-fold dilution) after gradient dilution were sequentially flowed over the chip surface from low concentration to high concentration at a flow rate of 30. Mu.l/min for a binding time of 180s and a dissociation time of 300s. Finally, the chip was regenerated using 10mM Glycine-HCl, pH 1.5 (BR-1003-54, cytiva).
The data results were analyzed by Biacore T200 analysis software (version number 3.1) using the 1:1 binding model.
FIG. 3B shows a representative affinity profile of binding of ADI-38497 PG antibodies and wild-type antibodies to anti-PG scFv fusion proteins using SPR. The results show that only the ADI-38497 PG antibody can specifically bind to the anti-PG scFv, which is the extracellular domain of the P329G CAR.
Table 6 SPR affinity of P329G BCMA antibodies and wild type antibodies to anti-PG scFv
(3-2) Detection of binding affinity of ADI-38497 molecule to anti-PG scFv fusion protein
Binding affinity of ADI-38497 molecules (P329G mutant antibodies or wild type antibodies) to anti-PG scFv fusion proteins was determined by Biacore T200. FIG. 3C shows a schematic representation of a method for determining antibody affinity (Avidity) using Surface Plasmon Resonance (SPR).
The specific method comprises the following steps: after coupling anti-Fab IgG (I5260, sigma) to the surface of HLC chip (HLC 30M, xantec), ADI-38497 molecules were captured on the chip surface and affinity and kinetic constants were obtained by detecting binding and dissociation of the chip surface antibody to the anti-PG scFv fusion protein in the mobile phase. The assay procedure used 10 XHBS-EP+ (BR-1006-69, cytiva) after 10-fold dilution as the assay buffer. Each cycle of affinity detection involved capture of ADI-38497 molecules, binding of one concentration of anti-PG scFv and chip regeneration. The anti-PG scFv after gradient dilution (concentration gradient 1.25nM-40nM, 2-fold dilution) was flowed over the chip surface at a flow rate of 30. Mu.l/min from low to high concentration in order of 180s for binding and 300s for dissociation. Finally, the chip was regenerated using 10mM glycine-HCl, pH 1.5 (BR-1003-54, cytiva).
The data results were analyzed by Biacore T200 analysis software (version number 3.1) using the 1:1 binding model.
FIG. 3D shows representative affinity profiles of ADI-38497 PG antibodies and wild-type antibodies with anti-PG scFv as the extracellular domain of P329G CAR using SPR assays. The results show that only the ADI-38497 PG antibody can specifically bind to P329G CAR.
Table 7 SPR affinity of P329G BCMA antibody and wild type antibody to anti-PG scFv
(3-3) Detection of P329G BCMA antibody and P329 CAR binding Activity
P329G CAR-T cells prepared in example 1 were resuscitated, resuspended in RPMI 1640 medium containing 10% FBS, and incubated at 37 ℃ for 24 hours. ADI-38497 PG antibody and wild-type ADI-38497 WT antibody were prepared as 5-fold gradient dilutions of antibody solutions with FACS buffer, incubated with 1E5 CAR positive cells for 30min at 4℃respectively, washed with FACS buffer and incubated with Fc gamma fragment-specific APC goat anti-human IgG (Jackson ImmunoResearch, 109-136-098) for another 30min at 4 ℃. Antibodies bound to cells were detected using flow cytometry, APC channel MFI was analyzed, plotted on the X-axis with antibody concentration and on the Y-axis and EC50 was calculated for binding.
FIG. 3E shows the binding capacity of ADI-38497 WT antibodies and ADI-38497 PG antibodies to P329G CAR-T cells. The results showed that only the P329G mutant antibody showed binding to CAR, whereas the WT antibody did not bind to CAR, consistent with the SPR results.
Table 8 summarizes the EC50 and EC90 values for binding of ADI-38497 PG and WT antibodies to P329G CAR-T cells of different donor origin.
Table 8 EC50 and EC90 values for binding of BCMA IgG antibodies to CAR-T cells
Example 4 detection of Fc Domain function of BCMA-specific P329G mutant antibodies
(4-1) ADI-38497 PG antibody ADCC Effect function detection
PBMC cells (PERIPHERAL BLOOD MONONUCLEAR CELLS ) from donor 3 were resuscitated, resuspended in RPMI 1640-containing medium, and stabilized at 37 ℃ for 1-2 hours. PBMCs and target cells were mixed at an effective target ratio of 25:1 and mixed with BCMA antibodies at different concentrations, incubated at 37 ℃ for 4 and 24 hours, respectively, and antibody-mediated killing of PBMCs against target cells was detected with LDH detection kit (Promega, G1780) and plotted and analyzed with the antibody concentration as X-axis and the cell lysis ratio as Y-axis. Cells were collected simultaneously, washed 2 times with FACS buffer, and CD3, CD56, CD16 and CD107a antibodies were added, wherein CD107a antibodies were added in advance and incubated with cells for 1 hour at 37 ℃. The above cell antibody mixture was stained at 4℃for 30 minutes, washed twice, resuspended in FACS buffer and detected by flow cytometry.
FIG. 4A shows the ability of ADI-38497 WT antibody and ADI-38497 PG antibody to mediate ADCC killing, which indicates that when tested for different incubation times (4 hours, 24 hours) and using different detection indicators (effects on cytotoxicity of target cells, on CD3, CD56, CD16 and CD107a expression), only WT antibody mediated ADCC cytotoxic injury to positive H929 tumor cells expressing BCMA, whereas P329G mutant antibodies lacked the ability to induce ADCC effect.
(4-2) ADCP Effect function detection of ADI-38497 PG antibody
ADCP reporter cell lines (Promega, G9871) in log phase were mixed with H929 cells at an effective target ratio of 2:1, 5:1, and mixed with BCMA antibodies at various concentrations, incubated for an additional 20 hours at 37℃and antibody-mediated reporter cell activation effects dependent on target cells were detected with a luciferase assay kit (Promega, E2620) and plotted and analyzed with changes in antibody concentration on the X-axis and fiber light reading on the Y-axis.
FIG. 4B shows the ability of ADI-38497 WT antibodies and ADI-38497 PG antibodies to mediate ADCP killing. The results indicate that when tested at different potency target ratios (2:1 or 5:1), all showed that only the ADI-38497 WT antibody mediated ADCP killing by BCMA expressing positive H929 tumor cells, whereas the P329G mutant antibody lacked the ability to induce ADCP killing by BCMA expressing positive H929 tumor cells.
(4-3) Functional assay with ADI-38497 PG antibody alone whether target cell lysis is mediated
Taking H929 cells and L363 cells in logarithmic growth phase, and paving the cells in a pore plate according to a certain quantity; and a part of the logarithmic phase H929 cells and L363 cells as target cells were subjected to mitomycin C treatment, to serve as a negative control. Subsequently, ADI-38497 PG antibodies were added at different concentrations and mixed, and incubation was continued at 37℃for 48 hours, 72 hours and 120 hours, respectively, the proportion of viable cells was measured using CellTiter-Glo (Promega, G9242), plotted and analyzed with co-incubation time on the X-axis and rayon light reading on the Y-axis.
FIG. 4C shows the ability of ADI-38497 PG antibody to mediate target cell lysis, and the results indicate that the ability to induce target cell lysis was lacking with ADI-38497 PG antibody alone when tested for different incubation times (48 hours, 72 hours, and 120 hours) and different ADI-38497 PG antibody concentrations (5 μg/ml, 50 μg/ml).
Example 5, P329G CAR-T in vitro function Studies
(5-1) CAR-T cell activation detection
The CAR-T cells prepared by donor 5 in example 1 were resuscitated and cultured overnight at 37 ℃. H929 or L363 tumor target cells and CAR-T cells were mixed according to E:T of 2:1, and 5-fold or 10-fold gradient diluted antibody solutions of different concentrations of ADI-38497 PG were added to a total volume of 200. Mu.L, and after incubation at 37℃for about 24 hours, the cells were collected by centrifugation, washed 2 times with FACS buffer, resuspended, added with FACS buffer containing LIVE/DEAD Fixable DEAD CELL STAIN, biotin-F (ab')2 Fragment goat anti-human IgG (Jackson ImmunoResearch, 109-066-006), stained for 30 minutes at 4℃and washed twice, CD4, CD8, CD25, CD69 and APC-strepitavidin antibody combinations were added, and the above cell antibody mixtures were stained for 30 minutes at 4℃and washed twice, resuspended with FACS buffer, and detected with a flow cytometer.
Figure 5A shows that only ADI-38497 PG antibodies containing the P329G mutation against H929 cells specifically mediate activation of CAR+ -T, up-regulate the expression levels of CD25, CD69, and activate indifferently on CD4+CAR+ and CD8+CAR+ cells, and exhibit ADI-38497 PG antibody concentration gradient dependence.
FIG. 5B shows the effect of different BCMA antibodies (ADI-38497 PG antibody, ADI-38497 WT antibody, GSK PG IgG) on the activation of HuR968B CAR-T cells by induced L363 target cells. As can be seen from fig. 5B, BCMA antibodies containing P329G mutation (ADI-38497 PG antibody, GSK PG IgG) were able to specifically mediate activation of car+ T cells, significantly up-regulating the expression levels of CD25, CD 69. There was also a weak up-regulation of CD25, CD69 expression in the CAR negative cell population, indicating a lower level of activation, but this degree of activation was not significant and negligible.
Thus, only BCMA antibodies containing P329G mutation (ADI-38497 PG antibody, GSK PG IgG) were able to specifically mediate activation of CAR+ -T, significantly up-regulate the expression levels of CD25, CD69, and have a P329G mutation BCMA antibody concentration gradient dependence, both in the case of high BCMA expressing H929 cells (fig. 5A) and in the case of low BCMA expressing L363 cells (fig. 5B) as target cells.
(5-2) CAR-T cell proliferation assay
The UNT cells and CAR-T cells prepared by donor 4 in example 1 were resuscitated and cultured overnight at 37 ℃. ADI-38497 WT antibody or ADI-38497 PG antibody was diluted with PBS, added to 96-well plates and incubated overnight at 4℃to coat the plates. CAR-T cells recovered by overnight culture were added to 96-well plates coated with antibodies as positive controls, and CD3/CD 28-coupled magnetic beads (magnetic beads: cell ratio 3:1) were directly added to CAR-T cells; incubation was carried out at 37℃for 72 hours and 120 hours, respectively, using CellTiter-Luminescent Cell Viability Assay (Promega, G7572) to detect the luminescence of the cells.
FIG. 5C shows proliferation of HuR968B CAR-T cells stimulated with coated ADI-38497 WT antibody or ADI-38497 PG antibody. The HuR968B CAR-T cells proliferate under stimulation with the coated ADI-38497 p329g antibody, and the ADI-38497 PG antibody proliferated the HuR968B CAR-T cells about 7-fold or 43.5-fold, respectively, after 3 days or 5 days of stimulation, as compared to stimulation with the ADI-38497 WT antibody; whereas UNT cells stimulated with ADI-38497 PG antibody, stimulation with ADI-38497 PG antibody did not result in significant proliferation of UNT cells compared to ADI-38497 WT antibody. Both HuR968B CAR-T cells and UNT cells proliferate significantly under stimulation with CD3/CD28 antibody coupled magnetic beads.
(5-3) Cytokine detection
The CAR-T cells prepared in example 1 were resuscitated and cultured overnight at 37 ℃. Tumor target cells and CAR-T cells were mixed at E:T of 2:1, and 5-fold or 10-fold gradient dilutions of different concentrations of BCMA antibody (ADI-38497 PG antibody, ADI-38497 WT antibody, or GSK PG IgG as Benchmark) were added to a total volume of 200. Mu.L, and after incubation at 37℃for about 24 hours, the supernatant was centrifuged, and collected. Cytokines were detected using BDTM Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II ((BD, 551809)). After mixing the Capture Beads in the kit in equal volumes, the plates were plated at 25. Mu.L/well. Equal volumes of supernatant or supernatant dilutions or standards were added. After mixing, 25. Mu.L of an equal volume of human Th1/Th2 PE detection reagent was added and incubated at room temperature for 3 hours in the dark. After washing twice with washing buffer, the suspension was resuspended and examined using a flow cytometer, and cytokine concentrations were calculated from the MFI values of the PE channels.
Fig. 5D shows the release of CAR-T cells secreting effector cytokines from donor 4 of example 1 following co-culture with H929 cells, RPMI8226 cells, after addition of different concentrations of BCMA antibody (ADI-38497 PG antibody, ADI-38497 WT antibody, or GSK PG IgG as positive control).
FIG. 5E shows the results of release of CAR-T cell-secreting effector cytokines after addition of different concentrations of BCMA antibody (ADI-38497 PG antibody, ADI-38497 WT antibody, or Benchmark GSK PG IgG) by co-culturing HuR968B CAR-T cells from Donor 5 (purchased from ORiCELLS, cat NO.: FPB004F-C, lot NO.: PCH20210100004, donor ID: Z0086) with different tumor cells.
As can be seen from fig. 5D and 5E, the HuR968B CAR-T cells were not activated in the case of addition of WT BCMA antibody (ADI-38497 WT antibody), did not secrete IL-2, IFN- γ, tnfα equivalent cytokines, and only in the case of addition of BCMA antibody containing P329G mutation (ADI-38497 PG antibody, or GSK PG IgG as positive control), the HuR968B CAR-T cells were activated, secreting effector cytokines. And according to the cytokine secretion results under different target cell conditions, the level of the cytokine secreted by the effector cells has no obvious correlation with the BCMA expression level of the target cells.
(5-4) Detection of killing efficiency
The CAR-T cells prepared in example 1 were resuscitated and cultured overnight at 37 ℃. Tumor target cells and CAR-T cells were mixed at E: T of 2:1 and 10-fold gradient dilutions of different concentrations of BCMA antibody (ADI-38497 PG antibody, ADI-38497 WT antibody, or Benchmark GSK PG IgG) were added to a total volume of 200 μl, incubated at 37 ℃ for about 24 hours, centrifuged, and the cell supernatants were transferred to 96-well Bai De elisa plates. LDH values in supernatants were measured using CytoTox 96Non-Radioactive Cytotoxicity Assay (Promega, G1780) and a microplate reader (Molecular Devices, spectromax i3 x) to calculate killing efficiency.
Fig. 5F shows the killing effect of different BCMA antibodies (ADI-38497 PG antibody, ADI-38497 WT antibody, or Benchmark GSK PG IgG) to induce HuR968B CAR-T cells on tumor cells (H929++ cells, RPMI 8236+++ cells, AMO1+ cells, and L363+ cells) with different BCMA expression levels. In the case of WT BCMA antibody addition, huR968B cells were not activated, did not produce a killing effect on tumor cells, and only in the case of P329G mutant-containing BCMA antibody addition, P329G CAR-T cells were activated, thereby killing target cells, and had an antibody concentration dependence, consistent with the CAR-T cell activation and effector cytokine results described above in this example. In addition, in the case of using H929, RPMI8226 cells that highly express BCMA as target cells, P329G BCMA antibodies mediated HuR968B CAR-T cells with higher killing effect than AMO1, L363 target cells that low-express BCMA.
Fig. 5G shows that neither P329G BCMA antibody nor WT BCMA antibody mediates the killing effect of CAR-T on BCMA negative target cells (e.g. BCMA-KO-H929 cells), nor on BCMA-expressing H929 cells (H929), nor on WT BCMA antibody, only P329G BCMA antibody can induce CAR-T to exert killing effect on tumor cells.
TABLE 9 EC50 values for the killing effect of P329G BCMA antibodies mediated HuR968B CAR-T cells on H929 target cells
| Antibodies to | EC50(nM) |
| ADI-38497 PG | 0.3502 To 0.7433 |
| ADI-38497 WT | Very wide |
Example 6 influence of free BCMA protein on PG CAR-T function
(6-1) Detection of killing efficiency
The CAR-T cells prepared by donor 6 in example 1 were resuscitated and cultured overnight at 37 ℃. Tumor target cells and CAR-T cells are mixed according to E:T of 2:1, PG BCMA antibody with a certain concentration is added, and BCMA antibody is not needed to be added for traditional Blue21 CAR-T. Different concentration BCMA protein solutions, each 10-fold diluted in gradient, were added as free BCMA protein to a total volume of 200 μl, incubated at 37 ℃ for about 24 hours, centrifuged, and the cell supernatants were transferred to 96-well Bai De elisa plates. LDH values in supernatants were measured using CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, G1780) and a microplate reader (Molecular Devices, spectromax i3 x) to calculate killing efficiency.
Figure 6 shows the effect of different concentrations of free BCMA protein on the HuR968B CAR-T and Blue21 CAR-T cell killing effect. In the case of the addition of different concentrations of free BCMA protein, once the conventional CAR T cells bind to the free BCMA protein, their function is blocked (due to the high affinity of conventional CAR molecules for BCMA), while PG CAR-T is still able to exert a normal killing effect when PG BCMA antibodies bind to soluble BCMA. Thus, the advantage of PG CAR-T cells is that their function is less affected by soluble ligands (e.g., soluble BCMA).
In vivo pharmacokinetic study of ADI-38497 PG antibodies, example 7
(7-1) Antibody injection and sampling
BALB/c mice (age 4-6 weeks, body weight 15-17g, female) were divided into 3 groups, namely ADI-38497 PG antibody, 1mg/kg antibody group; ADI-38497 PG antibody, 10mg/kg antibody panel; and ADI-38497 PG antibody, 200mg/kg antibody group, 9 mice per group; diluting the antibody to 0.1mg/mL,1mg/mL and 20mg/mL with 1 XPBS, wherein the administration volume of each mouse is 10mL/kg, namely the administration doses of the antibody are 1mg/kg,10mg/mL and 200mg/mL respectively; the administration mode is intravenous injection, and the administration frequency is single. 5 minutes, 30 minutes, 2 hours, 6 hours, 24 hours, 48 hours, 96 hours, 168 hours, 336 hours and 504 hours after antibody administration the mouse retroorbital venous plexus was collected with 100 μl of blood sample, centrifuged at 3000g, and the supernatant was aspirated for blood concentration determination.
(7-2) ADI-38497 PG antibody detection
And coating the 96-well ELISA plate one day in advance. The BCMA antigen was diluted to 1. Mu.g/mL with a coating solution (a powder of a carbonate (Thermo, 28382), dissolved in 400mL of ultra pure water, fixed to 500mL of volume, and mixed uniformly to give a coating solution), 100. Mu.L per well was sealed with a sealing plate, and the membrane was sealed at room temperature overnight. Pouring the coating solution, beating the coating solution on absorbent paper, adding 300 mu L of washing liquid into each hole, shaking and mixing for 10 seconds, beating the washing liquid, and repeating washing for 3 times. Adding sealing liquid into the gun, 200 mu L of sealing liquid is added into each hole, a sealing plate membrane sealing plate, incubate for 2h at room temperature. The plate was then washed 1 time. The diluted standard curve (prepared by gradient dilution of BCMA antibody of known concentration, standard curve (e.g. prepared by ADI-38497 PG antibody of known concentration) quality control sample and sample to be tested are incubated for 2h at room temperature per well 100 μl, the pre-coating solution is poured off, the solution is dried on absorbent paper, then 300 μl of wash solution is added per well, shaking and mixing for 10 seconds, after the wash solution is dried, washing is repeated 3 times.once again, goat anti-human IgG-Fc-HRP antibody (BETHYL) 1:10 ten-thousand dilution is repeated, 100 μl is added per well, room temperature is protected from light for 1h, followed by washing the plate 1 time, TMB substrate is added to 96-well ELISA plate, 100 μl per well is developed at room temperature for 5 min, 50 μl of stop solution is added per well, shaking is 10 seconds, OD450 nm and OD620 nm values are read within 30 minutes.
Figures 7A and 7B show the results of pharmacokinetic experiments with ADI-38497 PG antibody (hereinafter also referred to simply as PG Ab in vivo experiments in mice) in mice. The dose-dependent effect was shown by the exposure of ADI-38497 PG antibody (Cmax and AUClast) in serum after intravenous injection of 1mg/kg, 10mg/kg, 200mg/kg of ADI-38497 PG antibody, and no significant difference was observed in other pharmacokinetic parameters, as shown in Table 10, for ADI-38497 PG antibody 1mg/kg: AUC0-inf, cmax, CL, T1/2 is 2480. Mu.g×h/mL,30ug/mL,0.40mL/kg/h, 145h, respectively; ADI-38497 PG antibody 10mg/kg: AUC0-inf, cmax, CL, T1/2 is 24720. Mu.g×h/mL,187ug/mL,0.32mL/kg/h, 219h, respectively; ADI-38497 PG antibody 200mg/kg: AUC0-inf, cmax, CL, T1/2 were 397734. Mu.g.times.h/mL, 3895ug/mL,0.43mL/kg/h, 197h, ADI-38497 PG antibody 1mg/kg half-life slightly shorter than ADI-38497 PG antibody 10mg/kg and 200mg/kg, respectively.
TABLE 10 ADI-38497 PG pharmacokinetic experiment results
Example 8 effects of PG CAR-T cells in combination with different doses of PG antibody against bcmA highly expressing tumors in vivo
(8-1) Tumor inoculation and treatment of mice
H929 cells were resuspended in1 XPBS and prepared as a cell suspension at a cell concentration of 5X 106 cells/mL. NOG mice (age 4-6 weeks, body weight 15-17g, female) were shaved on the right back, subcutaneously injected with H929 cell suspension at a volume of 0.2 mL/mouse, i.e. an inoculum size of 1 x 106 cells/mouse. Mice with tumor volumes of 50.82-104.36 mm3 were divided into 7 groups, namely a PBS vehicle group, a PG Ab group, a PG CAR-T only group, a traditional CAR-T group, a PG Ab+PG CAR-T, a 3mg/kg antibody group, a PG Ab+PG CAR-T, a 1mg/kg antibody group and a PG Ab+PG CAR-T,0.3mg/kg antibody group, 7 mice per group, 7 days after tumor cell inoculation. Antibodies were prepared at concentrations of 0.3mg/mL,0.1mg/mL and 0.03mg/mL, and after completion of the grouping, antibody administration was performed on day 7, with a volume of 10mL/kg per mouse, with a frequency of 1 time per week, and the administration was intraperitoneal injection. The CAR-T cells prepared in donor 4 were resuspended in1 x PBS to prepare a cell suspension of 25 x 106 cells/mL for CAR+ cells and 0.2 mL/mouse was injected tail vein at day 7, i.e. 5 x 106 cells/mouse were reinfused for CAR+ cells. Mice body weight, tumor tissue maximum axis (L) and maximum axis (W) were monitored 2 times per week.
Figure 8A shows the therapeutic effect of different doses of PG antibody in combination with PG CAR-T cells in immunodeficient tumor-bearing mice subcutaneously vaccinated with human H929 highly expressing BCMA tumor cells. The results show that in BCMA high expression tumor model, administration of only PG CAR-T cells produced no significant anti-tumor effect, administration of only PG antibodies produced a certain anti-tumor effect, and only mice receiving both PG CAR-T cells and PG antibodies treated produced significant anti-tumor effect and exhibited antibody dose-dependent effects. Treatment of PG CAR-T cells with 0.3mg/kg antibody, 1mg/kg antibody and 3mg/kg antibody resulted in Tumor Growth Inhibition (TGI) of 92%, 88% and 101%, respectively, whereas the anti-tumor efficacy TGI in mice treated with PG CAR-T cells alone or PG antibodies alone was 25% and 53%, respectively. In addition, the anti-tumor effect generated by the PG CAR-T cell combined PG antibody of 3mg/kg is equivalent to that of the traditional Blue21 CAR-T cell with the same administration dosage, and the TGI is 101% and 102% respectively.
Figure 8B shows the weight change in mice in this experiment. The results showed that mice treated with PG CAR-T cells combined with PG antibody maintained stable body weight after receiving treatment, and increased body weight by 5.2%, 3.0%, 7.6% on average after treatment with PG antibody of 0.3mg/kg, 1mg/kg and 3 mg/kg. The result shows that the PG CAR-T cell combined PG antibody treatment produces obvious anti-tumor effect without obvious toxic and side effects.
(8-2) CAR-T cell detection in mice:
Taking 30 μl of the mouse blood sample of example 8-1, adding to a 96-well V-well plate, labeled as a sample detection well; mu.L of mouse blood was sampled and added to a 96-well V-well plate, labeled control well. 100. Mu.L of FACS buffer containing LIVE/DEAD Fixable DEAD CELL STAIN and TruStain FcXTM (anti-mouse CD 16/32) (Biolegend) was added to all wells, gently mixed and incubated at 4℃for 15 min in the absence of light; then, biotin-F (ab')2 Fragment goat anti-human IgG antibody is added into the sample detection hole, and the mixture is incubated for 30 minutes at 4 ℃ in a dark place; subsequently, 100. Mu.L of FACS buffer was added to each sample detection well, centrifuged at 400g, and the supernatant was discarded; to each well 100. Mu.L of FACS buffer containing APC-Cy7 anti-human CD45 (Biolegend), perCP-Cy5.5-CD3 (BD Biosciences) and APC-streptavidin (Biolegend) was added, gently mixed and incubated at 4℃for 30min in the absence of light; then 200. Mu.L/well FACS buffer was added to all wells, centrifuged at 400g and the supernatant discarded; add 250. Mu.L/well 1 XRBC Lysis/Fixation solution (Biolegend), mix well, incubate at room temperature for 20 minutes in the dark; centrifuging at 400g, and discarding the supernatant; after resuspension of the cells with 100 μl FACS buffer, 10 μl 123count ebeads was added per well and detected using a flow cytometer.
FIG. 8C shows the expansion of PG CAR-T cells in mice in the experiment of example 8-1. The results show that the in vivo expansion of PG CAR-T cells depends on PG antibodies, and shows a certain antibody dose dependence, and the mice in the group with higher doses have higher levels of PG CAR-T cell expansion. Only PG CAR-T cells were administered, which began to expand 1 week in reinfusion mice, expand to 466 cells/100 μl of peripheral blood after 2 weeks, reach higher levels (3644 cells/100 μl of peripheral blood) after 3 weeks, and remained high (3214 cells/100 μl of peripheral blood) after 4 weeks; in the case of different doses of PG antibody of 0.3mg/kg, 1mg/kg, 3mg/kg when PG CAR-T cells were combined, the peak in vivo expansion levels of PG CAR-T cells reached 11428 cells/100. Mu.L of peripheral blood, 19299 cells/100. Mu.L of peripheral blood, 35368 cells/100. Mu.L of peripheral blood after 2 weeks, respectively, which remained at higher levels after 4 weeks, 15486 cells/100. Mu.L of peripheral blood, 25073 cells/100. Mu.L of peripheral blood and 27666 cells/100. Mu.L of peripheral blood, respectively, far higher than the groups without co-administration of antibodies at the same time. In addition, conventional Blue21 CAR-T cells as positive controls showed similar kinetics of expansion, again starting to expand 1 week in reinfusion mice, reaching peak levels (174769 cells/100 μl peripheral blood) after 2 weeks, and remained at extremely high levels (131963 cells/100 μl peripheral blood) after 4 weeks.
Example 9 effects of PG CAR-T cells in combination with different doses of PG antibody against BCMA-underexpressing tumors in vivo
(9-1) Tumor vaccination and treatment of mice
L363 cells were resuspended in 1 XPBS to give a cell suspension with a cell concentration of 5X 106 cells/mL. NOG mice (age 4-6 weeks, body weight 15-17g, female) were shaved on the right back, injected subcutaneously with L363 cell suspension at a volume of 0.2 mL/mouse, i.e. an inoculum size of 1 x106 cells/mouse. Mice with tumor volumes of 74.14-110.29 mm3 were divided into 7 groups, namely a vehicle group, a PG Ab group, a PG CAR-T group, a traditional CAR-T group, a PG Ab+PG CAR-T group, a 3mg/kg antibody group, a PG Ab+PG CAR-T group, a 1mg/kg antibody group and a PG Ab+PG CAR-T group, and 7 mice per group, 9 days after tumor cell inoculation. Antibodies were prepared at concentrations of 0.3mg/mL,0.1mg/mL and 0.03mg/mL, and after completion of the grouping, antibody administration was performed on day 9, with a volume of 10mL/kg per mouse, and the frequency of administration was 1 time per week, and the administration mode was intraperitoneal injection. The CAR-T cells prepared in donor 4 were resuspended in 1 x PBS to prepare a cell suspension of 25 x106 cells/mL for CAR+ cells and 0.2 mL/mouse was injected tail vein at day 9, i.e. 5x 106 cells/mouse were reinfused for CAR+ cells. Mice body weight, tumor tissue maximum axis (L) and maximum axis (W) were monitored 2 times per week.
Fig. 9A shows the therapeutic effect of different doses of PG antibody in combination with PG CAR-T cells in immunodeficient tumor-bearing mice subcutaneously vaccinated with human L363 low expressing BCMA tumor cells. The results show that in BCMA low expressing tumor model, administration of PG CAR-T alone produced no anti-tumor effect, whereas administration of PG antibody alone produced no significant anti-tumor effect, TGI was 21%, and only mice receiving both PG CAR-T cells and PG antibody treatment produced significant anti-tumor effect, and exhibited antibody dose-dependent effects. In the case of PG CAR-T cells combined with PG antibody at a dose of 0.3mg/kg, PG CAR-T cells induced a significant anti-tumor effect, with TGI of 87%, when the combination was increased to PG antibody doses of 1mg/kg and 3mg/kg, the maximum anti-tumor effect induced by PG CAR-T cells was significantly increased, with TGI of 103% and 103%, respectively, showing the same anti-tumor effect as conventional Blue21 CAR-T cells.
Figure 9B shows the weight change in mice in this experiment. The results showed that mice treated with PG CAR-T cells combined with PG antibody had their body weights steadily increased after receiving treatment, and the average body weights increased by 18.2%, 10.5% and 8.5% after the combined PG antibodies of 0.3mg/kg, 1mg/kg and 3 mg/kg. The results indicate that PG CAR-T cell combined PG antibody treatment produced significant anti-tumor effects and did not induce significant toxicity.
(9-2) CAR-T cell detection in mice: the procedure is as in example 8-2.
FIG. 9C shows the expansion of PG CAR-T cells in mice in the experiment of example 9-1. The results show that administration of only PG CAR-T cells, which began to expand 1 week in the reinfusion mice, expanded to 919 cells per 100 μl of peripheral blood after 2 weeks, and rapidly declined to 204 cells per 100 μl of peripheral blood at 3 weeks; in the case of different doses of PG antibody of 0.3mg/kg, 1mg/kg and 3mg/kg when PG CAR-T cells were combined, the peak in vivo expansion level of PG CAR-T cells reached 4380 cells/100. Mu.L of peripheral blood, 8049 cells/100. Mu.L of peripheral blood and 3347 cells/100. Mu.L of peripheral blood after 2 weeks, which remained at higher levels after 3 weeks, 2475 cells/100. Mu.L of peripheral blood, 4121 cells/100. Mu.L of peripheral blood and 1969 cells/100. Mu.L of peripheral blood, respectively, far higher than that of the antibody group without co-administration at the same time. In addition, conventional Blue21 CAR-T cells as positive controls also expanded to peak levels (76836 cells/100 μl of peripheral blood) after 2 weeks in reinfusion mice, remaining at high levels (36328 cells/100 μl of peripheral blood) after 3 weeks.
Example 10 anti-tumor effects of different doses of PG CAR-T cells in combination with PG antibodies in vivo
(10-1) Tumor vaccination and treatment of mice
H929 cells were resuspended in 1 XPBS and prepared as a cell suspension at a cell concentration of 5X 106 cells/mL. NOG mice (age 4-6 weeks, body weight 15-17g, female) were shaved on the right back, subcutaneously injected with H929 cell suspension at a volume of 0.2 mL/mouse, i.e. an inoculum size of 1x106 cells/mouse. Mice with tumor volumes of 59.50-105.82 mm3 were divided into 7 groups, vehicle group, PG Ab group, PG CAR-T group alone, PG Ab+PG CAR-T group, 10×106 group, PG Ab+PG CAR-T group, 1×106 cell groups, PG Ab+PG CAR-T group, 0.1×106 cell groups and PG Ab+PG CAR-T group, 0.01×106 cell groups, 7 mice per group, 9 days after tumor cell inoculation. The antibody was prepared at a concentration of 0.3mg/mL, and after completion of the group, the antibody was administered on day 9, with a volume of 10mL/kg per mouse, at a frequency of 1 time per week, by intraperitoneal injection. The CAR-T cells prepared in donor 4 were resuspended in 1 XPBS to prepare a 50X 106/mL cell suspension of CAR+ cells, followed by 10-fold gradient dilution to prepare 5X 106、0.5×106 and 0.05X 106/mL cell suspensions, and the cell suspensions were injected at day 9 in tail vein at 0.2 mL/L. Mice body weight, tumor tissue maximum axis (L) and maximum axis (W) were monitored 2 times per week.
Figure 10A shows the therapeutic effect of PG antibodies in combination with different doses of PG CAR-T cells in immunodeficient tumor-bearing mice subcutaneously vaccinated with human H929 tumor cells. The results show that CAR-T cells produced similar anti-tumor effects to administration of PG antibody alone, with TGI of 49% and 50%, respectively, at very low doses of 0.01x106 CAR-T cells. Increasing the dose of CAR-T cells to 0.1x106、1×106、10×106 CAR-T cells, the anti-tumor effect induced by PG CAR-T cells was significantly increased, with TGI of 91%, 104%, 103% respectively. Administration of CAR-T cells alone did not show anti-tumor effects.
(10-2) CAR-T cell detection in vivo: the procedure is as in example 8-2.
FIG. 10B shows the expansion of PG CAR-T cells in mice in the experiment of example 10-1. The results showed that CAR-T cell reinfusion mice began to expand in vivo under PG antibody induction for 1 week, reached peak levels after 2 weeks, remained high after 3 weeks, and that CAR-T cell in vivo expansion was dependent on CAR-T cell dose, and that higher CAR-T dose group mice had higher levels of CAR-T cell expansion, with peak expansion levels of 0.01x106、0.1×106、1×106、10×106 CAR-T cell dose group of 6 cells per 100 μl peripheral blood, 338 cells per 100 μl peripheral blood, 3640 cells per 100 μl peripheral blood, 12895 cells per 100 μl peripheral blood, respectively, when PG antibody was used in combination.
Example 11 in vivo anti-tumor Effect study of PG CAR-T cells in combination with PG antibodies at different dosing frequencies
(11-1) Tumor inoculation and treatment of mice
H929 cells were resuspended in1 XPBS and prepared as a cell suspension at a cell concentration of 5X 106 cells/mL. NOG mice (age 4-6 weeks, body weight 15-17g, female) were shaved on the right back, subcutaneously injected with H929 cell suspension at a volume of 0.2 mL/mouse, i.e. an inoculum size of 1x106 cells/mouse. Mice with tumor volumes of 51.25-94.97 mm3 were divided into 7 groups, vehicle group, PG CAR-T-only group, PG Ab+PG CAR-T, Q3/4X 2 group, PG Ab+PG CAR-T, QW X4 group, PG Ab+PG CAR-T, Q2W X2 group, PG Ab+PG CAR-T, Q3W X2 group and traditional CAR-T group, 7 mice per group, 7 days after tumor cell inoculation. The PG Ab antibody with the concentration of 0.1mg/mL is prepared, and after the grouping is completed, the antibody is administrated, and the administration volume of each mouse is 10mL/kg, and the administration mode is intraperitoneal injection. The CAR-T cells prepared in donor 4 were resuspended in 1x PBS and prepared as 10 x106 cells/mL CAR+ cells/mL cell suspension, and 0.2 mL/tail vein injection of cell suspension. Mice body weight, tumor tissue maximum axis (L) and maximum axis (W) were monitored 2 times per week.
FIG. 11A shows the frequency of administration of PG antibody in the experiment of example 11-1. Q3-4D 2: the PG antibody was administered 2 times at week 1 with a 3-4 day interval on a 4 week cycle; q3w×2: the PG antibody was administered 2 times at a frequency of 3 weeks/time; q2w×2: the PG antibody was administered 2 times at a frequency of 2 weeks/time; qw×4: the PG antibody was administered 4 times at a frequency of 1 week/time.
Figure 11B shows the therapeutic effect of PG antibody at different dosing frequency when PG CAR-T cells are combined in immunodeficient tumor bearing mice subcutaneously vaccinated with human H929 tumor cells. The results show that when the PG CAR-T cells are combined, the PG antibodies can induce the PG CAR-T cells to generate obvious anti-tumor effect at different dosing frequencies, and the dosing frequency of the PG antibodies is increased, so that the anti-tumor effect induced by the PG CAR-T cells is obviously increased, the anti-tumor effect induced by the QW multiplied by 4 CAR-T cells is most obvious, and the anti-tumor effect similar to that of the traditional Blue21 CAR-T cells is shown.
(11-2) CAR-T cell detection in vivo: the procedure is as in example 8-2.
FIG. 11C shows the expansion of PG CAR-T cells in mice in the experiment of example 11-1. The results show that when PG CAR-T cells are combined, CAR-T cell expansion kinetics are similar at different dosing frequencies of PG antibodies, CAR-T cell reinfusion into mice begins to expand for 1 week, reaches peak levels after 2 weeks, and drops to baseline levels after 3 weeks. In addition, traditional Blue21 CAR-T cells also proliferated to peak levels after 2 weeks in reinfusion mice, and remained at high levels after 3 weeks.
Example 12 in vivo study of systemic tumor Effect of PG CAR-T cells in combination with different doses of PG antibody
(12-1) Tumor inoculation and treatment of mice
First, H929-luc cells were prepared. Specifically, a lentivirus containing GFP-luciferase gene was packaged with H929 cells (available from Nanjac, bai Biotechnology Co., ltd.) and the H929 cells were infected with the obtained lentivirus, followed by sorting by flow cytometry to obtain a GFP-luciferase double-expressed H929-luc cell line.
H929-luc cells were then resuspended in 1 XPBS to a cell concentration of 25X 106 cells/mL. NOG mice (age 4-6 weeks, body weight 15-17g, female) were injected with H929-luc cell suspension in a volume of 0.2 mL/mouse tail vein. 14 days after tumor cell inoculation, substrate D-Luciferin (15 mg/mL) was injected intraperitoneally, at a volume of 10 mL/kg/mouse, and analyzed by IVIS select imaging 10 minutes after substrate injection. Mice with a rayon optical signal at 1.17X107～1.43×108 photons/sec were divided into 7 groups, vehicle group, PG Ab,0.3mg/kg group, PG Ab,3mg/kg group, PG CAR-T group, PG Ab+PG CAR-T,0.3 mg/kg+2X106 group, PG Ab+PG CAR-T,3 mg/kg+2X106 group and Blue21 CAR-T group, each group being 6-7 mice. Antibodies were prepared at concentrations of 0.03mg/mL and 0.3mg/mL, respectively, and antibody administration was started on day 14 after completion of the group, with a volume of 10mL/kg per mouse, and with a frequency of 1 time per week, by intraperitoneal injection. The CAR-T cells prepared in donor 4 were resuspended in 1x PBS to prepare 10 x106 cells/mL of CAR+ cells suspension, and 0.2 mL/mL of cell suspension was injected into the tail vein on day 7.
Figure 12A shows fiber light images of the therapeutic effect of different doses of PG antibody in combination with PG CAR-T cells in immunodeficient tumor-bearing mice vaccinated with human H929-luc tumor cells in the tail vein. The results show that in the systemic tumor model, PG antibody and PG CAR-T cells also generate significant anti-tumor effect, on the 18 th day, the light intensity of the rayon light distribution and the light intensity of the mice with PG antibody of 0.3mg/kg and PG CAR-T cells are significantly reduced compared with those of the control group, meanwhile, the mice with PG antibody of 3mg/kg and PG CAR-T cells have no obvious light signal, and the traditional Blue21 CAR-T treatment group still has a large amount of light distribution, so that the PG CAR-T cells induce the anti-tumor effect faster than Blue21 CAR-T; the PG antibody 3mg/kg combined PG CAR-T cell group mice had no apparent rayon optical signal maintained until day 56, until day 63, and part of the mice tumors showed slightly rayon optical signal, while at this time more than half of the mice tumors of the Blue21 CAR-T treated group had apparent recurrence and metastasis to the peritoneal cavity, indicating that the PG CAR-T cell combined PG antibody produced a more durable anti-tumor effect.
Figure 12B shows the therapeutic effect of different doses of PG antibody in combination with PG CAR-T cells in immunodeficient tumor-bearing mice vaccinated with human H929-luc tumor cells in the tail vein. The results show that under the condition of 0.3mg/kg of PG antibody, PG CAR-T cells generate a certain anti-tumor effect, the PG antibody dosage is increased to 3mg/kg, the anti-tumor effect induced by PG CAR-T cells is obviously increased, the anti-tumor effect is maintained longer, the PG antibody is stopped to be administered, the tumor growth starts to relapse about 4 weeks, and the traditional Blue21 CAR-T tumor to which the same cell dosage is administered relapse faster, so that the PG CAR-T cells have longer-lasting anti-tumor effect than the traditional Blue21 CAR-T. The administration of the PG CAR-T cell group alone, the PG antibody group did not show significant anti-tumor effects.
Fig. 12C shows the change in mouse body weight in the above experiment. The results showed a smooth rise in body weight for each treatment group during treatment, suggesting that different doses of PG antibody in combination with PG CAR-T cell treatment did not induce significant toxicity in the hematological tumor model.
Example 13 in vivo toxicity study of PG CAR-T cells in combination with PG antibodies
(13-1) Tumor vaccination and treatment of mice
H929 cells were resuspended in 1 XPBS and prepared as a cell suspension at a cell concentration of 5X 106 cells/mL. NOG mice (age 4-6 weeks, body weight 15-17g, female) were shaved on the right back, subcutaneously injected with 5 x 106/mL H929 cell suspension, and injected in a volume of 0.2 mL/mouse. 6 days after tumor cell inoculation, mice with tumor volumes of 38.49-104.77 mm3 are divided into 8 groups, namely a carrier group without tumor, PG Ab+PG CAR-T,10 mg/kg+10X106 group respectively as shown in Table 11; and tumor-bearing vehicle groups, PG CAR-T groups, PG Ab groups, PG Ab+PG CAR-T,10mg/kg+10×106 groups, PG Ab+PG CAR-T,3mg/kg+10×106 groups, and PG Ab+PG CAR-T,3mg/kg+1×106 groups, 24 mice per group. Antibodies with the concentration of 1mg/mL and 0.3mg/mL are respectively prepared, and after grouping, the antibodies are administered, the administration volume of each mouse is 10mL/kg, the administration frequency is 1 time per week, the administration times are 3 times, and the administration mode is intraperitoneal injection. 1 day after antibody administration, CAR-T cells prepared by donor 4 were resuspended with 1 x PBS, and CAR+ cell suspensions were prepared at 50 x 106 and 5 x 106/mL, respectively. The tail vein was injected with 0.2 mL/cell suspension. Mice body weight, tumor tissue maximum axis (L) and maximum axis (W) were monitored 2 times per week. Peripheral blood was taken before antibody 1, after CAR-T back infusion, before antibody 3 and at the end of the experiment, 4 mice per group, for hematological and blood biochemical tests.
Table 11 treatment and dosing of groups
Figure 13A shows the therapeutic effect of PG antibodies in combination with PG CAR-T cells in immunodeficient tumor-bearing mice subcutaneously vaccinated with human H929 tumor cells. The results show that the PG antibody combined with the PG CAR-T cell treatment group all generate significant anti-tumor effect, while the mice with the PG antibody treatment group only have weaker anti-tumor effect, and only PG CAR-T does not generate anti-tumor effect.
Fig. 13B shows the change in mouse body weight in this experiment. The results show that the mice in the tumor-bearing and non-tumor-bearing treatment groups have a smooth increase in body weight and no significant change in body weight compared to control mice, suggesting that different doses of PG antibody in combination with different doses of PG CAR-T cell therapy did not induce significant toxic responses.
Fig. 13C and 13D show the results of the hematological and blood biochemical tests in the mice in the above experiments. The results show that the hematological and blood biochemical indexes of the tumor-bearing and non-tumor-bearing treated mice are not obviously changed compared with the control mice during the treatment period, which indicates that the PG antibody combined with PG CAR-T cell treatment does not produce toxic reaction.
EXAMPLE 14 clinical Studies
1. Test drugs and other drugs:
The active components of the test drug described in this example are: P329G BCMA antibody (ADI-38497 PG antibody) comprising the heavy chain variable region shown in SEQ ID NO:2 and the heavy chain constant region shown in SEQ ID NO:5, and the light chain variable region shown in SEQ ID NO:3 and the light chain constant region shown in SEQ ID NO: 6) and P329G CAR-T cells (patient autologous PBMC cells were transformed to obtain CAR-T cells).
The preparation of P329G BCMA antibody was prescribed at 20.0mg/ml of P329G BCMA antibody, 0.76mg/ml of histidine,
1.08Mg/ml histidine hydrochloride, 50.00mg/ml sorbitol, 0.20mg/ml polysorbate 80, pH 6.0; specification of P329GBCMA antibody: 60mg (3 mL)/bottle.
Specification of P329G CAR-T cells: each bag contains 90-140X 106 anti-P329G Fc fragment CAR positive cells dissolved in 5% -7.5% DMSO cryoprotectant; the manufacturing company for P329G CAR-T cells is Xinda cell pharmaceutical (Suzhou) Co.
The pharmaceutical combination comprising ADI-38497 PG antibody and P329G CAR-T cells is hereafter referred to as BCMA-CAR-PG.
2. Group in/out subject criteria
Criteria for inclusion
1) 18 Years old and older, with unlimited sex.
2) According to the diagnostic criteria for multiple myeloma of the international working group for myeloma (International Myeloma Working Group, IMWG), there are test evidence that multiple myeloma was first diagnosed.
3) At least 3 anti-myeloma treatment regimens (past treatments comprising proteasome inhibitors and immunomodulators) were followed, wherein induction chemotherapy, stem cell transplantation and maintenance treatments administered sequentially were considered 1 treatment regimen if no disease progression occurred in the middle of treatment; disease progression (according to IMWG criteria) as evidenced by the examination data occurs during or within 12 months after the recent anti-myeloma treatment.
4) The presence of measurable lesions upon screening was determined according to any of the following criteria:
① Bone marrow cytology, bone marrow biopsy tissue or flow detection monoclonal plasma cell proportion is more than or equal to 5%;
② The level of the clone protein (M-protein) of the blood list is more than or equal to 0.5g/dL;
③ The urine M protein level is more than or equal to 200mg/24 hours;
④ Light chain multiple myeloma without measurable lesions in serum or urine: serum immunoglobulin free light chain not less than 10
Mg/dL and serum immunoglobulin kappa/lambda free light chain ratio are abnormal.
5) ECOG scores 0 or 1.
6) The expected survival time is more than or equal to 12 weeks.
Exclusion criteria:
1) With graft-versus-host disease (GVHD), or in need of immunosuppressants.
2) Patients who received autologous hematopoietic stem cell transplantation (autologous stem-cell transplantation, ASCT) or who had previously received allogeneic hematopoietic stem cell transplantation (allogeneic hematopoietic stem cell transplantation, allo-HSCT) within 12 weeks prior to PBMC collection.
3) No non-mobilized mononuclear cells can be harvested for CAR-T cell production.
4) The following antitumor treatments have been previously accepted:
① The cytotoxic or proteasome inhibitor therapy, or, within 14 days prior to the time that the mononuclear cells can be harvested;
② Systemic steroid therapy was being received within 7 days before mononuclear cells could be harvested, or;
③ Treatment of multiple myeloma with monoclonal antibodies within 21 days prior to single nuclear cell harvesting, or;
④ Receiving an immunomodulatory treatment within 7 days prior to the time that the mononuclear cells can be harvested, or;
⑤ Targeted therapy, epigenetic therapy, or invasive trial medical device use, or within 14 days prior to the single nuclear cell harvest;
⑥ Radiation therapy was received within the first 14 days of the single nucleated cells could be harvested. But if the portal covers less than or equal to 5% of the bone marrow reserves, then
No matter what day the radiation therapy was over, the subject was eligible to participate in the study, or;
⑦ White blood cell apheresis was accepted in the pre-7 single nuclear cell apheresis.
5) Subjects who were judged by the investigator to require chronic systemic steroid treatment during the treatment period (except for inhaled or topical use, dose < 10 mg/day).
6) A patient with hypertension (blood pressure is 140/90 mmHg) with a history of hypertension and uncontrollable by drug treatment.
7) Severe heart disease: including but not limited to unstable angina, myocardial infarction (within 6 months prior to screening), congestive heart failure (New York Heart disease Association [ New York Heart Association, NYHA ] class. Gtoreq.III), severe arrhythmias.
8) Unstable systemic disease was judged by researchers: including but not limited to severe liver, kidney or metabolic diseases requiring drug therapy.
9) Malignant tumors other than multiple myeloma were present within 5 years prior to screening, except for fully treated cervical carcinoma in situ, basal cell or squamous cell skin carcinoma, and post-radical treatment breast ductal carcinoma in situ.
10 A history of solid organ transplants.
11A history of T cell mediated-based autoimmune disease (e.g.: type 1 diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, etc.), if a pre-group investigator judges that the above-mentioned autoimmune disease indication mainly mediated by T cells may exist, the related diseases need to be detected and excluded from the group.
12 A patient suspected of or having symptoms of a plasma cell tumor central nervous system violation.
13 A stroke or convulsive episode occurred within 6 months prior to signing the ICF.
14 Screening for plasma cell leukemia (according to domestic standards, the plasma cells in the peripheral blood leukocyte classification are greater than 20% or the absolute value of the plasma cells is greater than or equal to 2.0X109/L), fahrenheit macroglobulinemia, POEMS syndrome (polyneuropathy, visceral enlargement, endocrinopathy, monoclonal proteinopathy and skin changes) or primary systemic immunoglobulin light chain amyloidosis (SYSTEMIC LIGHT-chain amyloidosis, AL).
15 Live attenuated vaccine was vaccinated within 4 weeks prior to single nuclear cell collection.
16 Large surgery within 2 weeks prior to mononuclear cell harvesting, or surgery planned to occur within 2 weeks after single harvesting or after first administration of study treatment (note: subjects scheduled for surgery for local anesthesia may participate in the study).
17 Is participating in other interventional clinical studies when signing ICFs.
18 7 Days prior to the acquisition of mononuclear cells, there are active or uncontrollable infections (CTCAE) requiring systemic treatment
Except for grade 1 genitourinary infections and upper respiratory tract infections).
3. Administration method and process
After the subjects in the group receive peripheral blood mononuclear cells (PERIPHERAL BLOOD MONONUCLEAR CELL, PBMC) for single harvest (D-33-D-26 (-33 days to-26 days)), P329G CAR-T cell preparations (hereinafter referred to as P329G CAR-T cells) are prepared by using the T cells of the subjects themselves. After successful preparation of P329G CAR-T cells, the subject will first receive a clear shower pretreatment regimen of cyclophosphamide and fludarabine (D-5-D-3 (-5 to-3 days)); infusion of 1 dose of P329G BCMA antibody (also known as ADI-38497 PG antibody) was received two days after the end of pretreatment and resting; P329G BCMA antibody was infused within 1-2 hours after infusion was completed or the next day (D2) received P329G CAR-T cell infusion; thereafter, from the day of P329GBCMA antibody infusion, each 21 days was followed by a cycle of periodic infusion of ADI-38497 PG (the first cycle (days 1-21) comprised both QW x 3 administration and Q3W administration, the second and subsequent Q3W administration) until disease progression, intolerance, withdrawal of informed consent, or other reasons for cessation of study treatment (based on the pre-emergence).
The relevant dosing regimen is shown in the following table:
Dose escalation phase:
In the dose escalation phase, the classical "3+3" dose escalation mode was used. ADI-38497 PG antibodies total 4 dose groups: 0.1mg/kg, 0.3mg/kg, 1mg/kg, 3mg/kg; P329G CAR-T cells total 2 dose groups: 2 x 106/kg and 5 x 106/kg. First, the optimal dose of ADI-38497 PG was fully explored with a fixed P329G CAR-T cell dose, starting with 2 x 106/kg P329G CAR-T cells and 0.1mg/kg ADI-38497 PG, and if the starting dose was intolerant, the cell dose was considered to fall back to 0.75 x 106/kg. The DLT window was 21 days after the initial dose of ADI-38497 PG antibody, if the above BCMA-CAR-PG dose did not reach the maximum tolerizing dose (maximum tolerated dose, MTD), the ADI-38497 PG antibody dose was fixed and the P329G CAR-T cell dose was increased to 5 x 106/kg to continue the exploration.
During the up-dosing phase, the possible dose groups for BCMA-CAR-PG administration were as follows:
Dose group 1:0.1mg/kg ADI-38497 PG antibody+2 x 106/kg P329G CAR-T cells (n=3-6);
dose group 2:0.3mg/kg ADI-38497 PG antibody+2 x 106/kg P329G CAR-T cells (n=3-6);
Dose group 3:1mg/kg ADI-38497 PG antibody+2×106/kg P329G CAR-T cells (n=3-6);
dose group 4:3mg/kg ADI-38497 PG antibody+2×106/kg P329G CAR-T cells (n=3-6);
Dose group 5:3mg/kg ADI-38497 PG antibody+5×6/kg P329G CAR-T cells (n=3-6)
Exploring the dose group: such as: 1mg/kg ADI-38497 PG antibody +5 x 106/kg P329G CAR-T cells (n=3-6) or based on prior observations, an exploratory dose set was selected after full discussion by the investigator and sponsor.
The relevant dosing amounts of P329G CAR-T cells and P329G BCMA antibodies are shown in the following table:
4. experimental results
The index meaning of the experimental result is as follows
1. Curative effect:
The C-BCMA-CAR-PG-01 scheme and the C-BCMA-CAR-PG-02 scheme are summarized as follows:
C-BCMA-CAR-PG-Y001: group 6 subjects were enrolled, with 0.3mg/kg dose group 3, 1mg/kg dose group 3. The best tumor evaluation result of 3 subjects is PR after the evaluation of researchers. Of these, the 0.3mg/kg dose group had one case where the best tumor evaluation result of the subject reached PR and disease progression occurred after 7 months of duration; two subjects in the 1mg/kg dose group reached PR as a result of optimal tumor assessment, one disease progression after 2 months of duration (as shown in FIG. 14).
C-BCMA-CAR-PG-Y002: group 5 subjects were enrolled, with 0.1mg/kg dose group 1, 0.3mg/kg dose group 3, 1mg/kg dose group 1. The study was evaluated that the 0.1mg/kg dose group 1 subjects reached the best tumor assessment result VGPR (very good partial remission) 3 months after feedback, lasting VGPR in the last 6 month visit. The 0.3mg/kg dose group had 1 subject reached the best tumor evaluation result PR at the last visit of 3 months (as shown in FIG. 15).
2. Cell PK:
C-BCMA-CAR-PG-01: median values (range) of peak time to cell expansion (Tmax) for the 0.3mg/kg (n=3) and 1mg/kg (n=3) dose groups were 20 (14-33.8) day and 13.8 (9.71-26.8) day, respectively; the geometric mean (coefficient of variation) of the peak cell expansion (Cmax) is 1.01 x 10≡4 (148.6%) and 10.3 x 10≡4 (50.6%) copies/. Mu.g DNA, respectively; the geometric mean (coefficient of variation) of the cells AUC0-21d is 4.24 x 10≡4 (145%) and 17.4 x 10≡4 (89.4%) day copies/. Mu.g DNA, respectively.
C-BCMA-CAR-PG-02: median values (ranges) of cell expansion peak times (Tmax) for the 0.1mg/kg (n=1), 0.3mg/kg (n=3) and 1mg/kg (n=1) dose groups were 20.9day,16 (12.9-17.9) day and 20.9day, respectively; the geometric mean (coefficient of variation) of the peak cell expansion (Cmax) is 7 x 10≡4 (NaN), 3.27 x 10≡4 (85.1%) and 1.66 x 10≡4 (NaN) copies/. Mu.g DNA, respectively; the geometric mean (coefficient of variation) of the cells AUC0-21d is 47.1 x 10≡4 (NaN), 19.1 x 10≡4 (69.4%) and NaN (NaN) day copies/. Mu.g DNA, respectively.
3. Antibody PK
C-BCMA-CAR-PG-01: geometric means (coefficient of variation) of peak drug concentrations (Cmax) for the 0.3mg/kg (n=3) and 1mg/kg (n=3) dose groups were 4.88 (97.2%) and 9.89 (71.9%) μg/mL, respectively; the geometric mean (coefficient of variation) of the area under the plasma concentration-time curve (AUC 0-last) was 200 (132.4) and 117.6 (127%) μg.h/mL, respectively; the geometric mean value (variation coefficient) of the clearance rate (CL) is NaN (NaN) and 0.000671 (104.9%) L/h/kg respectively; the geometric mean (coefficient of variation) of half-life (t 1/2) was NaN (NaN) and 88.9 (133.6%) h, respectively. Within the dosage range of 0.3-1 mg/kg, target-mediated drug elimination dominates, and ADI-38497 PG antibody is rapidly cleared.
C-BCMA-CAR-PG-02: geometric mean (coefficient of variation) of peak drug concentrations (Cmax) for 0.1mg/kg (n=1), 0.3mg/kg (n=3) and 1mg/kg (n=1) dose groups were 1.95 (NaN), 3.99 (58.1%) and 7.83 (NaN) μg/mL, respectively; the geometric mean (coefficient of variation) of the area under the plasma concentration-time curve (AUC 0-last) was 20.2 (NaN), 23.8 (82.4%) and 30 (NaN) μg.h/mL, respectively; the geometric mean (coefficient of variation) of the Clearance (CL) is 0.00439 (NaN), naN (NaN) and NaN (NaN) L/h/kg respectively; the geometric mean (coefficient of variation) of half-life (t 1/2) was 9.79 (NaN), naN (NaN) and NaN (NaN) h, respectively. Within the dosage range of 0.1-1 mg/kg, target-mediated drug elimination dominates, and ADI-38497 PG antibody is rapidly cleared.
At the present doses, the target-mediated drug elimination effect is evident, suggesting that the frequency of administration of the antibody with QW in the first cycle will be more beneficial for maintenance of effective antibody concentration.
4. Security data:
In the C-BCMA-CAR-PG-01 study, there were 5 subjects with adverse events related to study drug (TREATMENT RELATED ADVERSE EVENT, TRAE), the most frequent TRAE being a decrease in neutrophil count (5, 83.3%), a decrease in lymphocyte count (5, 83.3%), a decrease in white blood cell count (5, 83.3%) and a decrease in platelet count (5, 83.3%).
In the C-BCMA-CAR-PG-02 study, there were 5 subjects with TRAE, the most frequent TRAE being a decrease in neutrophil count (5, 100%), a decrease in white blood cell count (5, 100%), a decrease in platelet count (5, 100%) and anemia (5, 100%).
The results of the non-clinical research, the action mechanism, the clinical research experience of the traditional similar medicines and the preliminary clinical safety data are combined, so that the product of the invention achieves better effect on safety:
(1) CRS: by the date of data expiration, in two studies, 5 out of 11 subjects developed CRS with varying degrees, the CRS incidence was 45.5% lower than that of existing BCMA-CART products, in the existing case, the CRS that had developed was relatively controllable, and the subjects had improved or healed after treatment;
(2) ICANS (immune effector cell-related neurotoxic syndrome): by the date of data expiration, both studies were performed and no neurological toxic events were seen. Overall, BCMA-CAR-PG has good safety and tolerability, CRS and ICANS occur at lower rates than existing products.
(3) And (3) clinical results show that CART cells in a subject to which the product is applied can be maintained for a longer time, and the method has a certain guiding significance on clinical treatment effects.
EXAMPLE 15 typical case
Typical case 1: C-BCMA-CAR-PG-02 subjects, women, age 46, igG-kappa multiple myeloma, DS stage III, without extramedullary lesions, received 7 previous chemotherapy regimens containing proteasome inhibitors, immunomodulators, glucocorticoids. The proportion of bone marrow plasma cells at the baseline stage is 31%. Patients were signed informed consent at 2022.6.16 to enter the antibody 0.1mg/kg, cell 2 x106/kg dose group. 2022.6.27, single nuclear cell collection, 2022.8.9, first antibody and cell feedback, 2022.8.16 and 8.23 respectively, in a first cycle, second and third antibody administration; second and third cycle antibody administration is performed at 2022.8.30, 2022.9.20; missed fourth and fifth cycle dosing for epidemic reasons; 2022.11.28 to a sixth cycle; the seventh period and the eighth period are missed for administration due to epidemic reasons; 2023.2.3 and 2022.2.23 were subjected to the ninth cycle, and the tenth cycle was administered. Patients had a tumor evaluation of PR for 1 month, a tumor evaluation of VGPR for 3 months and 6 months, and continued remission. The results of patient cell PK are shown in FIG. 18 below, D15 reached the peak of amplification at 105 copies/ug DNA, patient CAR-T was better in persistence, CAR-T was also detected half a year after reinfusion, and CAR-T was also detected at 103 copies/ug DNA patient's apparent peak of cytokines including IL-6, IL-10, IL-2R, TNF-alpha at about 10 days after reinfusion, and then dropped to the normal range at about 15 days. Patients develop grade 1 CRS on day 9 after reinfusion for 6 days of post-remission.
Typical case 2: C-BCMA-CAR-PG-01 subject, male, 43 years old, was kappa light chain type multiple myeloma, DS stage III-A, combined with extramedullary lesions. The proportion of bone marrow plasma cells at the baseline stage is 0.5%. Patients were signed informed consent at 2022.4.27 and entered into the antibody 0.3mg/kg, cell 2x 106/kg dose group. 2022.5.11 single nuclear cell collection, 2022.6.20 first antibody infusion, 2022.6.21 cell reinfusion, followed by antibody dosing every three weeks for a total of 9 cycles of antibody dosing. The tumor evaluation result of the patients 3 weeks after administration was SD, the efficacy evaluation 9 weeks after administration was PR, and PR continued for 7 months after disease progression. The results of the patient's cell PK are shown in FIG. 20 below, D15 reached an amplification peak at a level of 105 copies/ug DNA, and the patient had seen a clear peak in cytokines including IL-6, IL-10, TNF- α about 10 days after cell reinfusion, followed by a decrease to the normal range of values about 15 days (FIG. 21).
While exemplary embodiments of the invention have been described above, it will be appreciated by those skilled in the art that these disclosures are exemplary only, and that various other substitutions, adaptations, and modifications may be made within the scope of the invention. Therefore, the present invention is not limited to the specific embodiments set forth herein.
Exemplary sequence