Drawings
FIG. 1 is a bar graph representing the profile of CD84 (alias LY9B, SLAMF, hCD84, mCD 84) gene expression in all tumor samples and paired normal tissues. The height of the bars represents the median expression of a certain tumor type or normal tissue. From http:// gepia. Cancer-pku. Cn/detail. Phenyl=cd84.
FIG. 2 is a box plot (10-90 percentile) of CD84mRNA expression in cell lines obtained from different tumor types as measured by RNA sequencing (top) or Affymetrix microarray (bottom). From https:// portals. The figure shows only the 20 tumor types with highest CD84 expression.
Expression of CD84 in different cell lines. The light gray histogram represents staining with isotype matched control antibody and the dark gray histogram represents staining with specific CD84 antibody.
FIG. 4. CD84 expression in samples of nine patients diagnosed with chronic lymphocytic leukemia were assessed by flow cytometry. The light gray histogram represents staining with isotype matched control antibody and the dark gray histogram represents staining with specific CD84 antibody.
Fig. 5. CD84 expression in samples of ten patients diagnosed with acute myeloid leukemia were assessed by flow cytometry. One representative example patient P04 for medium-expressed CD84 is shown, and one representative example patient P10 for high-expressed CD 84. The light gray histogram represents staining with isotype matched control antibody and the dark gray histogram represents staining with specific CD84 antibody.
FIG. 6 is a diagram of the pCCL-EF 1. Alpha. -CAR 84 plasmid vector showing the location (above) where the CD84CAR sequence has been inserted. Scheme for anti-CD 84 second generation CAR constructs (bottom).
FIG. 7.24h after incubation, IFN- γ secretion by CD 84-targeted CAR-T cells co-cultured with Ramos cells at a 2:1 effector to target ratio. UT: non-transduced T cells. Statistical significance was determined with Kruskal-Wallis (multiple comparisons with UT). Average + -SEM for 4 experiments is shown. * P <0.001, p <0.01, p <0.05.
FIG. 8.24h incubation, IL-2 secretion by CD 84-targeted CART cells co-cultured with Ramos cells at a 2:1 effector to target ratio. UT: non-transduced T cells. Statistical significance was determined with Kruskal-Wallis (multiple comparisons with UT). Average + -SEM for 4 experiments is shown. * P <0.001, p <0.01, p <0.05.
FIG. 9.24h incubation followed by granzyme B secretion from CD 84-targeted CART cells co-cultured with Ramos cells at a 2:1 effector to target ratio. UT: non-transduced T cells. Statistical significance was determined with Kruskal-Wallis (multiple comparisons with UT). Average + -SEM for 4 experiments is shown. * P <0.001, p <0.01, p <0.05.
FIG. 10.24h incubation, TNF- α secretion by CD 84-targeted CART cells co-cultured with Ramos cells at a 2:1 effector to target ratio. UT: non-transduced T cells. Statistical significance was determined with Kruskal-Wallis (multiple comparisons with UT). Average + -SEM for 4 experiments is shown. * P <0.001, p <0.01, p <0.05.
FIG. 11 cytotoxicity assays of different CD 84-targeted CART cells versus Ramos-GFP+ cells after a 24 hour incubation period at a 2:1 effector to target cell ratio. The percentage of target surviving cells relative to untreated cells (target cells only) is shown. Mean ± SEM of 8 independent experiments. UT: non-transduced T cells. Statistical significance was determined using a common one-way ANOVA (multiple comparisons with UT): * P <0.001, p <0.01.
FIG. 12 cytotoxicity assays of different CD84 targeted CART cells versus K562-GFP+ cells after a 24 hour incubation period at a 2:1 effector to target cell ratio. The percentage of target surviving cells relative to untreated cells (target cells only) is shown. Mean ± SEM of 8 independent experiments. UT: non-transduced T cells. Statistical significance was determined using a common one-way ANOVA (multiple comparisons with UT): * P <0.001, p <0.01.
FIG. 13 cytotoxicity assays of different CD84 targeted CART cells versus Ramos-GFP+ cells after 24 and 48 hours incubation periods at 4:1, 2:1, 1:1 and 0.5:1 effector to target (E: T) ratios. The percentage of target surviving cells relative to untreated cells (target cells only) is shown. Average ± SEM of 5 independent experiments. UT: non-transduced T cells. Statistical significance was determined using a common one-way ANOVA (multiple comparisons with UT): * P <0.001, p <0.05.
FIG. 14 cytotoxicity assays of different CD84 targeted CART cells versus K562-GFP+ cells after 24 and 48 hours incubation periods at 4:1, 2:1, 1:1 and 0.5:1 effector to target (E: T) ratios. The percentage of target surviving cells relative to untreated cells (target cells only) is shown. UT: non-transduced T cells. Average ± SEM of 5 independent experiments. Statistical significance was determined using a common one-way ANOVA (multiple comparisons with UT): * P <0.01, p <0.05, n.s.: is not significant.
FIG. 15 cytotoxicity assays of different CD84 targeted CART cells versus MOLM-13-GFP+ cells after 24 and 48 hour incubation periods at 4:1, 2:1, 1:1 and 0.5:1 effector to target cell (E: T) ratios. The percentage of target surviving cells relative to untreated cells (target cells only) is shown. Average ± SEM, UT of 3 independent experiments: non-transduced T cells. Statistical significance was determined using a common one-way ANOVA (multiple comparisons with UT): * P <0.001, p <0.01, p <0.05.
FIG. 16 cytotoxicity assays of different CD84 targeted CART cells versus NALM6-GFP+ cells after 24 and 48 hours incubation periods at 4:1, 2:1, 1:1 and 0.5:1 effector to target (E: T) ratios. The percentage of target surviving cells relative to untreated cells (target cells only) is shown. Average ± SEM of 3 independent experiments. UT: non-transduced T cells. Statistical significance was determined using a common one-way ANOVA (multiple comparisons with UT): * P <0.001, p <0.01, p <0.05.
FIG. 17 cytotoxicity assays of different CD84 targeted CART cells versus MOLT4-GFP+ cells at 4:1, 2:1, 1:1 and 0.5:1 effector to target cell ratios after 24 and 48 hours incubation periods. The percentage of target surviving cells relative to untreated cells (target cells only) is shown. Average ± SEM of 4 independent experiments. UT: non-transduced T cells. Statistical significance was determined using a common one-way ANOVA (multiple comparisons with UT): * P <0.01, p <0.05, ns: is not significant.
Fig. 18. Expression of CD84 in Peripheral Blood Mononuclear Cells (PBMCs) was assessed by flow cytometry. The light gray histogram represents staining with isotype matched control antibody and the dark gray histogram represents staining with specific CD84 antibody. PBMCs were stained with specific antibodies to CD4, CD8, CD19 and CD 14.
Figure 19.16 hours incubation period, cytotoxicity assays were performed on different CD84 targeted CART cells with PBMCs labeled with CFSE+ and Ramos-GFP+ cells. The percentage of target surviving cells relative to untreated cells (target cells only) is shown. Average ± SEM of 3 independent experiments from 3 different donors. UT: non-transduced T cells. Statistical significance was determined by paired T-test (compared to UT): * P <0.01, n.s.=insignificant.
SDS PAGE gel image of the CD84 antigen. Reducing SDS PAGE stained with coomassie brilliant blue.
FIG. 21 CD84 expression on peripheral blood leukemia cells from two patients diagnosed with T-ALL (P01 and P02) was assessed by flow cytometry. Histograms represent CD84 expression on the parent cells for CD34 gating; the light grey histogram represents staining with isotype matched control antibody and the dark grey histogram represents staining with specific CD84 antibody.
Figure 22 CAR T cell expansion 6-7 days after transduction was shown as a fold increase relative to the number of T cells cultured on day 0. The figures represent 5 to 12 independent amplifications, each with cells from different healthy donors. UT: non-transduced T cells.
FIG. 23 cytotoxicity assays of different CD 84-targeted CART cells versus Ramos-GFPffLuc cells after 24 and 48 hours incubation periods at 4:1, 2:1, 1:1 and 0.5:1 effector to target (E: T) ratios. The percentage of target surviving cells relative to untreated cells (target cells only) is shown. Average ± SEM of 5 independent experiments. UT: non-transduced T cells. Statistical significance was determined using a two-way ANOVA test (multiple comparisons with UT): * P <0.001, p <0.01, p <0.05.
FIG. 24 cytotoxicity assays of different CD 84-targeted CART cells versus MOLM-13GFPffLuc cells after 24 and 48 hours incubation periods at 4:1, 2:1, 1:1 and 0.5:1 effector to target cell (E: T) ratios. The percentage of target surviving cells relative to untreated cells (target cells only) is shown. Average ± SEM of at least 5 independent experiments. UT: non-transduced T cells. Statistical significance was determined using a two-way ANOVA test (multiple comparisons with UT): * P <0.001, p <0.01, p <0.05.
FIG. 25 cytotoxicity assays of different CD84 targeted CART cells versus U937GFPffLuc cells after 24 and 48 hours incubation periods at 4:1, 2:1, 1:1 and 0.5:1 effector to target (E: T) ratios. The percentage of target surviving cells relative to untreated cells (target cells only) is shown. Average ± SEM of at least 3 independent experiments. UT: non-transduced T cells. Statistical significance was determined using a two-way ANOVA test (multiple comparisons with UT): * P <0.001, p <0.01, p <0.05.
FIG. 26 cytotoxicity assays of different CD84 targeted CART cells versus MOLT-4GFPffLuc cells after 24 and 48 hours incubation periods at 4:1, 2:1, 1:1 and 0.5:1 effector to target (E: T) ratios. The percentage of target surviving cells relative to untreated cells (target cells only) is shown. Average ± SEM of at least 5 independent experiments. UT: non-transduced T cells. Statistical significance was determined using a two-way ANOVA test (multiple comparisons with UT): * P <0.001, p <0.01, p <0.05.
FIG. 27 cytotoxicity assays of different CD84 targeted CART cells versus CFSE stained primary AML cells after 24 and 48 hours incubation periods at 4:1, 2:1, 1:1 and 0.5:1 effector to target cell (E: T) ratios. The percentage of target surviving cells relative to untreated cells (target cells only) is shown. UT: non-transduced T cells. Statistical significance was determined using a two-way ANOVA test (multiple comparisons with UT): * P <0.001, p <0.01, p <0.05.
FIG. 28 cytotoxicity assays of different CD84 targeted CART cells versus primary T-ALL cells stained with CFSE after 24 and 48 hours incubation periods at 4:1, 2:1, 1:1 and 0.5:1 effector to target cell (E: T) ratios. The percentage of target surviving cells relative to untreated cells (target cells only) is shown. UT: non-transduced T cells. Statistical significance was determined using a two-way ANOVA test (multiple comparisons with UT): * P <0.001, p <0.01, p <0.05.
FIG. 29.24h incubation followed by IFN- γ secretion by CD84 targeted CAR-T cells co-cultured with Ramos, MOLM-13 or MOLT-4 cells as indicated at a 2:1 effector to target ratio. UT: non-transduced T cells. Statistical analysis was determined using two-way ANOVA mixed effect analysis (assuming spherical) and Dunnet post test (for multiple comparisons) (as compared to UT). Average ± SD of at least 3 experiments are shown. * P <0.001, p <0.01, p <0.05.
FIG. 30.24h after incubation, granzyme B secretion of CD 84-targeted CAR-T cells by co-culture with Ramos, MOLM-13 or MOLT-4 cells as indicated at a 2:1 effector to target cell ratio. UT: non-transduced T cells. Statistical analysis was determined using two-way ANOVA mixed effect analysis (assuming spherical) and Dunnet post test (for multiple comparisons) (as compared to UT). Average ± SD of at least 3 experiments are shown. * P <0.001, p <0.01, p <0.05.
FIG. 31.24h after incubation, TNF- α secretion from CAR-T cells was targeted by CD84 co-cultured with Ramos, MOLM-13 or MOLT-4 cells as indicated at a 2:1 effector to target ratio. UT: non-transduced T cells. Statistical analysis was determined using two-way ANOVA mixed effect analysis (assuming spherical) and Dunnet post test (for multiple comparisons) (as compared to UT). Average ± SD of at least 3 experiments are shown. * P <0.001, p <0.01, p <0.05.
Figure 32 measurement of in vitro CART cell proliferation by CFSE assay at the 4 day time point (flow cytometry images). Proliferation on day 0, after 4 days of medium only (control), upon stimulation with IL-2 or in the presence of MOLM-13AML cells. UT: non-transduced T cells.
Figure 33 CD84, CD33 and CD123 expression on CD34+ HPSC isolated from apheresis products of healthy donors for allogeneic stem cell transplantation were assessed by flow cytometry. The light grey histogram represents staining with isotype matched control antibody and the dark grey histogram represents staining with specific antibody.
FIG. 34 cytotoxicity assay of different CD84 targeted CART cells with CD34+ HPSCs isolated from 5 different cord blood units after a 24 hour incubation period at 4:1 and 2:1 effector to target cell (E: T) ratios. The percentage of target surviving cells relative to untreated cells (target cells only) is shown. Average ± SEM of 5 independent experiments. UT: non-transduced T cells. Statistical significance was determined using two-way ANOVA (multiple comparisons with UT): * P <0.001, p <0.01, p <0.05.
FIG. 35 cytotoxicity assays of different CD84 targeted CART cells with CD34+ HPSCs isolated from apheresis products of healthy donors for allogeneic stem cell transplantation after an incubation period of 24 hours at 4:1, 2:1, 1:1 and 0.5:1 effector to target cell (E: T) ratios. The percentage of target surviving cells relative to untreated cells (target cells only) is shown. Average ± SEM of three independent experiments. UT: non-transduced T cells. Statistical significance was determined using two-way ANOVA (multiple comparisons with UT): * P <0.001, p <0.01, p <0.05.
FIG. 36 cytotoxicity assays of different CD84 targeted CART cells versus CD3+ T cells isolated from the same donor after 24 hours incubation period at 4:1, 2:1, 1:1 and 0.5:1 effector to target cell (E: T) ratios. The percentage of target surviving cells relative to untreated cells (target T cells only) is shown. Average ± SEM of 5 independent experiments. UT: non-transduced T cells. Statistical significance was determined using two-way ANOVA (multiple comparisons with UT): * P <0.001, p <0.01, p <0.05.
Fig. 37. Different T cell subsets on target T cells prior to co-culture with different CART84 and on target T cells surviving after co-culture with different CART84 were analyzed by flow cytometry. The following T cell subpopulations were studied: CD45RA+/CD62L+ primary cells (white), CD45RA-/CD62L+ central memory cells (dark grey), CD45RA-/CD 62L-effector memory cells (black) and CD45RA+/CD 62L-effector T cells (light grey).
FIG. 38 efficacy of CART84 cells against MOLM-13 cells in vivo. MOLM-13 disease progression was monitored weekly by bioluminescence. Bioluminescence quantification (a) and animal survival (B). The quantitative analysis of bioluminescence was performed using a two-way ANOVA model in comparison to Dunnett's multiplex comparison to UT treated mice. The Log-Rank test was used to determine statistical significance of survival with 3 comparisons of corrected p-values.
FIG. 39 efficacy of CART84 cells against MOLM-13 cells in vivo. MOLM-13 disease progression was monitored weekly by bioluminescence. Bioluminescence quantification (a) and animal survival (B). At the end of the experiment, the total number of MOLM-13GFP-ffLuc cells (C), CD3+ T cells (D) and CART cells (E) in the mouse bone marrow (upper) and spleen (lower) was assessed by flow cytometry. The quantitative analysis of bioluminescence was performed using a two-way ANOVA model in comparison to Dunnett's multiplex comparison to UT treated mice. The Log-Rank test was used to determine statistical significance of survival with 3 comparisons of corrected p-values. Statistical analysis of MOLM-13 and CD3+ T cells compared to UT treated mice using a one-way ANOVA model with Dunnett's multiple comparisons; and CART cells were quantitatively analyzed using a one-way ANOVA model with Tukey's multiple comparisons.
FIG. 40. Efficacy of CD84CART cells against MOLM-13 cells in vivo. The progression of MOLM-13 disease was followed once per week by bioluminescence. Bioluminescence quantification (a) and animal survival (B). At the end of the experiment, the total number of MOLM-13GFP-ffLuc cells (C), CD3+ T cells (D) and CART cells (E) in the mouse bone marrow (upper) and spleen (lower) was assessed by flow cytometry. The quantitative analysis of bioluminescence was performed using a two-way ANOVA model in comparison to Dunnett's multiplex comparison to UT treated mice. The Log-Rank test was used to determine statistical significance of survival with 3 comparisons of corrected p-values. Statistical analysis of MOLM-13 and CD3+ cells with one-way ANOVA model versus Dunnett's multiple comparisons versus UT treated mice; and CART cells were quantitatively analyzed using a one-way ANOVA model with Tukey's multiple comparisons.
FIG. 41. Efficacy of CD84CART cells against MOLT-4 cells in vivo. MOLT-4 disease progression was monitored weekly by bioluminescence. Bioluminescence quantification (a) and animal survival (B). The quantitative analysis of bioluminescence was performed using a two-way ANOVA model in comparison to Dunnett's multiplex comparison to UT treated mice. The Log-Rank test was used to determine statistical significance of survival with 3 comparisons of corrected p-values.
FIG. 42 efficacy of CD84CART cells against MOLT-4 cells in vivo. MOLT-4 disease progression was monitored weekly by bioluminescence. Bioluminescence quantification (a) and animal survival (B). At the end of the experiment, the total number of MOLT-4GFP-ffLuc cells (C), cd3+ T cells (D) and CART cells (E) in the mouse bone marrow (upper) and spleen (lower) were examined by flow cytometry. The quantitative analysis of bioluminescence was performed using a two-way ANOVA model in comparison to Dunnett's multiplex comparison to UT treated mice. The Log-Rank test was used to determine statistical significance of survival with 3 comparisons of corrected p-values. Statistical analysis of MOLT-4 and CD3+ cells compared to UT treated mice using a one-way ANOVA model with Dunnett's multiple comparisons; and CART cells were quantitatively analyzed using a one-way ANOVA model with Tukey's multiple comparisons.
FIG. 43 CD84 expression on human primary cells was assessed by flow cytometry. The light gray histogram represents staining with isotype matched control antibody and the dark gray histogram represents staining with specific CD84 antibody.
FIG. 44 cytotoxicity assays of CART84 cells 152.3, 153.5 and UT versus human primary cells using XCELLigence instrument measurements were performed 72 hours after CART/UT addition (arrow) to the cell culture. UT: non-transduced T cells.
FIG. 45 efficacy of CD84CART cells against Ramos cells in vivo. (A) Ramos disease progression was monitored weekly by bioluminescence. The quantitative analysis of bioluminescence was performed using a two-way ANOVA model in comparison to Dunnett's multiplex comparison to UT treated mice. (B) The total number of Ramos GFP-ffLuc cells in the mouse bone marrow at endpoint was assessed by flow cytometry. Statistical analysis was performed with one-way ANOVA model versus Dunnett's multiple comparisons versus UT treated mice. UT: non-transduced T cells. * p <0.05.
Detailed Description
As used herein, the terms "comprises" and "comprising" are synonymous with "comprising" or "including," and are inclusive or open-ended, and do not exclude additional, unrecited members, elements, or steps. The terms "comprising" and "comprised of" also include the term "consisting of.
CD84
CD84 (also known as LY9B and SLAMF 5) is a membrane glycoprotein that is a member of the Signaling Lymphocyte Activation Molecule (SLAM) family, which itself is a subset of the larger CD2 cell surface receptors in the Ig superfamily.
The extracellular portion of the CD84 receptor contains an atypical IgV distal domain and an IgC2g proximal domain, a structure common to all members of the SLAM family. CD84 functions as an homophilic adhesion molecule and is expressed in a variety of immune cell types. Receptor ligand interactions involve the IgV domain and are independent of the cytoplasmic domain. There are specific differences in homophilic interfaces that prevent CD84 from binding to other molecules of the SLAM family.
The inventors have determined that CD84 is overexpressed in a range of cell lines derived from hematological malignancies, including burkitt's lymphoma, acute Myeloid Leukemia (AML), chronic Myeloid Leukemia (CML), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), and histiocyte lymphoma.
Studies have shown that CD84 is overexpressed in Chronic Lymphocytic Leukemia (CLL).
Examples of the amino acid sequence of CD84 are:
MAQHHLWILLLCLQTWPEAAGKDSEIFTVNGILGESVTFPVNIQEPRQVKIIAWTSKTSVAYVTPGDSETAPVVTVTHRNYYERIHALGPNYNLVISDLRMEDAGDYKADINTQADPYTTTKRYNLQIYRRLGKPKITQSLMASVNSTCNVTLTCSVEKEEKNVTYNWSPLGEEGNVLQIFQTPEDQELTYTCTAQNPVSNNSDSISARQLCADIAMGFRTHHTGLLSVLAMFFLLVLILSSVFLFRLFKRRQGRIFPEGSCLNTFTKNPYAASKKTIYTYIMASRNTQPAESRIYDEILQSKVLPSKEEPVNTVYSEVQFADKMGKASTQDSKPPGTSSYEIVI
(SEQ ID NO:126)
Antigen binding domains
The antigen binding domain may be a protein or peptide having the ability to recognize and bind to an antigen. Antigen binding domains include any naturally occurring, synthetic, semisynthetic, or recombinantly produced binding partner for an antigen of interest.
Illustrative antigen binding domains include antibodies or antibody fragments or derivatives, extracellular domains of receptors (e.g., TCRs), cell surface molecule/receptor ligands, or receptor binding domains thereof.
In preferred embodiments, the antigen binding domain is or is derived from an antibody. An antibody-derived domain may be a fragment of an antibody or a genetically engineered product of one or more fragments of an antibody, which fragments are involved in binding to an antigen. Examples include variable regions (Fv), complementarity Determining Regions (CDRs), fab, single chain variable fragments (scFv), heavy chain variable regions (VH), light chain variable regions (VL), and camelidae antibodies (VHH).
The antigen binding domain may be non-human (e.g., murine), chimeric, humanized or fully human.
In a preferred embodiment, the antigen binding domain is a single chain variable fragment (scFv). For example, the scFv may be a murine scFv, a human scFv, or a humanized scFv.
In the case of antibodies or antigen binding fragments thereof, the term "complementarity determining regions" (CDRs) refers to highly variable loops in the heavy or light chain variable regions of the antibody. CDRs can interact with the antigen conformation and to a large extent determine binding to the antigen (although some framework regions are known to be involved in binding). The heavy chain variable region and the light chain variable region each contain 3 CDRs.
"Heavy chain variable region" (VH) refers to a variable fragment of an antibody heavy chain that contains three CDRs inserted between flanking extensions called framework regions, which are more highly conserved than the CDRs and form a scaffold that supports the CDRs.
"Light chain variable region" (VL) refers to a variable fragment of an antibody light chain that contains three CDRs inserted between framework regions.
"Fv" refers to the smallest fragment of an antibody that carries the complete antigen binding site. Fv consists of the variable region of a single light chain combined with the variable region of a single heavy chain.
"Single chain variable fragment" (scFv) refers to an engineered antibody consisting of a light chain variable region (VL) and a heavy chain variable region (VH) linked to each other directly or via a peptide linker sequence.
The antigen binding domain may specifically bind to an antigen, e.g., bind to an antigen but not to other peptides, or bind to other peptides with lower affinity.
The binding affinity between two molecules (e.g., antigen binding domain and antigen) can be quantified, for example, by determining the dissociation constant (KD). KD can be determined by measuring the kinetics of complex formation and dissociation between the antigen binding domain and the antigen, for example using techniques such as Surface Plasmon Resonance (SPR). The rate constants corresponding to association and dissociation of the complex are referred to as association rate constant Ka (or Kon) and dissociation rate constant Kd (or Koff).KD is associated with Ka and Kd by the following equation: KD＝kd/ka, respectively.
Suitably, the antigen binding domain is a CD84 binding domain.
In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having Complementarity Determining Regions (CDRs) with the following sequences: CDR1-NYWIN (SEQ ID NO: 1); CDR2-DIYPVSGTTNYNEKFKR (SEQ ID NO: 2); and CDR3-GTGRFAY (SEQ ID NO: 3); or variants thereof each having up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequence: CDR1-RASQSVSTSSYSYMH (SEQ ID NO: 4); CDR2-FASNLES (SEQ ID NO: 5); and CDR3-QHSWEIPYT (SEQ ID NO: 6); or variants thereof each having up to three amino acid substitutions, additions or deletions.
In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having Complementarity Determining Regions (CDRs) with the following sequences: CDR1-NYWLG (SEQ ID NO: 7); CDR2-DIYPGGGYTNYIEKFKG (SEQ ID NO: 8); and CDR3-YEGGYYGNYDAMDY (SEQ ID NO: 9), or variants thereof each having up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequence: CDR1-RASESVDNYGISFMN (SEQ ID NO: 10); CDR2-AASNQGS (SEQ ID NO: 11); and CDR3-QQSKAVPRT (SEQ ID NO: 12), or variants thereof each having up to three amino acid substitutions, additions or deletions.
In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having Complementarity Determining Regions (CDRs) with the following sequences: CDR1-GFTFSSYA (SEQ ID NO: 13); CDR2-ISGSGGST (SEQ ID NO: 14); and CDR3-AKWDCSDGRCYWAY (SEQ ID NO: 15), or variants thereof each having up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequence: CDR1-NIESKD (SEQ ID NO: 16); CDR2-DDA (SEQ ID NO: 17); and CDR3-QVWDSSSDHVV (SEQ ID NO: 18), or variants thereof each having up to three amino acid substitutions, additions or deletions.
In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having Complementarity Determining Regions (CDRs) with the following sequences: CDR1-GFTFSSYP (SEQ ID NO: 19); CDR2-ISYHGRNK (SEQ ID NO: 20); and CDR3-ARDRDATPGGTGVGNHGMAV (SEQ ID NO: 21), or variants thereof each having up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequence: CDR1-QSLLHSSGYNY (SEQ ID NO: 22); CDR2-MGS (SEQ ID NO: 23); and CDR3-MQGLQTPPT (SEQ ID NO: 24), or variants thereof each having up to three amino acid substitutions, additions or deletions.
In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having Complementarity Determining Regions (CDRs) with the following sequences: CDR1-GFTFSDNA (SEQ ID NO: 25); CDR2-ISGTGRTT (SEQ ID NO: 26); and CDR3-AKWDCSDGRCYWAY (SEQ ID NO: 27), or variants thereof each having up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequence: CDR1-QSLVYSDGDTY (SEQ ID NO: 28); CDR2-KVS (SEQ ID NO: 29); and CDR3-MQGTHWPPNT (SEQ ID NO: 30), or variants thereof each having up to three amino acid substitutions, additions or deletions.
In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having Complementarity Determining Regions (CDRs) with the following sequences: CDR1-TSGMGVG (SEQ ID NO: 31); CDR2-HIWWDDVKRYNPALKS (SEQ ID NO: 32); and CDR3-MRTSYYFDY (SEQ ID NO: 33), or variants thereof each having up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequence: CDR1-RASENIFSSLA (SEQ ID NO: 34); CDR2-NAKTLAE (SEQ ID NO: 35); and CDR3-QHHYATPFT (SEQ ID NO: 36), or variants thereof each having up to three amino acid substitutions, additions or deletions.
In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having Complementarity Determining Regions (CDRs) with the following sequences: CDR1-SYWIN (SEQ ID NO: 37); CDR2-DIYLGSGSTNYNEKFKS (SEQ ID NO: 38); and CDR3-SGGYLGY (SEQ ID NO: 39), or variants thereof each having up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequence: CDR1-RASQSVSTSSYSYMH (SEQ ID NO: 40); CDR2-FASNLES (SEQ ID NO: 41); and CDR3-QHSWEIPYT (SEQ ID NO: 42), or variants thereof each having up to three amino acid substitutions, additions or deletions.
In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having Complementarity Determining Regions (CDRs) with the following sequences: CDR1-NYWIG (SEQ ID NO: 43); CDR2-DIYPGGGYTNYNENFKG (SEQ ID NO: 44); and CDR3-STTYYSSYWCFDV (SEQ ID NO: 45), or variants thereof each having up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequence: CDR1-KSSQSLLNSGNQANYLA (SEQ ID NO: 46); CDR2-GASTRES (SEQ ID NO: 47); and CDR3-QNDHSYPFT (SEQ ID NO: 48), or variants thereof each having up to three amino acid substitutions, additions or deletions.
In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having Complementarity Determining Regions (CDRs) with the following sequences: CDR1-RYWMS (SEQ ID NO: 49); CDR2-EINPDSSTINYTPSLKD (SEQ ID NO: 50); and CDR3-PGPTVVATYWYFDV (SEQ ID NO: 51), or variants thereof each having up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequence: CDR1-RSSQSIVHSNGNTYLE (SEQ ID NO: 52); CDR2-KVSSRFS (SEQ ID NO: 53); and CDR3-FQGSHVPRT (SEQ ID NO: 54), or variants thereof each having up to three amino acid substitutions, additions or deletions.
In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having Complementarity Determining Regions (CDRs) with the following sequences: CDR1-RYWIN (SEQ ID NO: 55); CDR2-DIYPGSGSTNYNEKFKS (SEQ ID NO: 56); and CDR3-DTTIAY (SEQ ID NO: 57), or variants thereof each having up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequence: CDR1-RASQSVTTSRYSYMH (SEQ ID NO: 58); CDR2-FASNLES (SEQ ID NO: 59); and CDR3-QHSWEIPYT (SEQ ID NO: 60), or variants thereof each having up to three amino acid substitutions, additions or deletions.
In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having Complementarity Determining Regions (CDRs) with the following sequences: CDR1-GYNMN (SEQ ID NO: 131); CDR2-NIDPYYGGTNYNQKFKG (SEQ ID NO: 132); and CDR3-GLLSGSFPY (SEQ ID NO: 133), or variants thereof each having up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequence: CDR1-RASENIYSYLA (SEQ ID NO: 134); CDR2-NAKTLAE (SEQ ID NO: 135); and CDR3-QHHYGSPLT (SEQ ID NO: 136), or variants thereof each having up to three amino acid substitutions, additions or deletions.
In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having Complementarity Determining Regions (CDRs) with the following sequences: CDR1-RSWMS (SEQ ID NO: 137); CDR2-EINPDSSTINYTPSLKD (SEQ ID NO: 138); and CDR3-FYDGYSIYWYFDV (SEQ ID NO: 139), or variants thereof each having up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequence: CDR1-RSSQSIVHSNGDTYLE (SEQ ID NO: 140); CDR 2-KVSNSRFS (SEQ ID NO: 141); and CDR3-FQGSHVPRT (SEQ ID NO: 142), or variants thereof each having up to three amino acid substitutions, additions or deletions.
In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having Complementarity Determining Regions (CDRs) with the following sequences: CDR1-TSGMGVG (SEQ ID NO: 143); CDR2-HIWWDDVKRYNPALRS (SEQ ID NO: 144); and CDR3-IAVTYFFDF (SEQ ID NO: 145), or variants thereof each having up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequence: CDR1-RASENIFSSFA (SEQ ID NO: 146); CDR2-NARTLAE (SEQ ID NO: 147); and CDR3-QHHYASPFT (SEQ ID NO: 148), or variants thereof each having up to three amino acid substitutions, additions or deletions.
In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having Complementarity Determining Regions (CDRs) with the following sequences: CDR1-TSGMGVG (SEQ ID NO: 149); CDR2-HIWWDDVKRYNPALKS (SEQ ID NO: 150); and CDR3-MSTSYYFDY (SEQ ID NO: 151), or variants thereof each having up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequence: CDR1-KASQSLFTSVA (SEQ ID NO: 152); CDR2-SASYRYT (SEQ ID NO: 153); and CDR3-QQHYSSPFT (SEQ ID NO: 154), or variants thereof each having up to three amino acid substitutions, additions or deletions.
In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having Complementarity Determining Regions (CDRs) with the following sequences: CDR1-IYAMN (SEQ ID NO: 155); CDR2-RIRSKSNNYARFYADSVKD (SEQ ID NO: 156); and CDR3-PLRSYFSMDY (SEQ ID NO: 157), or variants thereof each having up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequence: CDR1-KASENVDTYVS (SEQ ID NO: 158); CDR2-GASNRYT (SEQ ID NO: 159); and CDR3-GQTYSYPWT (SEQ ID NO: 160), or variants thereof each having up to three amino acid substitutions, additions or deletions.
In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID No. 61; and a VL domain having the sequence of SEQ ID No. 62 or 108, or a variant thereof each having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID NO. 63; and a VL domain having the sequence of SEQ ID No. 64 or 109, or a variant thereof each having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID No. 65; and a VL domain having the sequence of SEQ ID No. 66, or variants thereof each having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID No. 68; and a VL domain having the sequence of SEQ ID No. 69, or variants thereof each having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID NO. 71; and a VL domain having the sequence of SEQ ID No. 72, or variants thereof each having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID NO. 74; and a VL domain having the sequence of SEQ ID No. 75, or a variant thereof each having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID NO. 76; and a VL domain having the sequence of SEQ ID NO 77, or variants thereof each having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID NO. 78; and a VL domain having the sequence of SEQ ID No. 79, or variants thereof each having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID No. 80; and a VL domain having the sequence of SEQ ID No. 81, or variants thereof each having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID NO. 82; and a VL domain having the sequence of SEQ ID No. 83, or variants thereof each having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID No. 161; and a VL domain having the sequence of SEQ ID No. 162, or variants thereof each having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID No. 163; and a VL domain having the sequence of SEQ ID No. 164, or variants thereof each having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID No. 165; and a VL domain having the sequence of SEQ ID NO 166, or variants thereof each having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID NO 167; and a VL domain having the sequence of SEQ ID No. 168, or variants thereof each having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID No. 169; and a VL domain having the sequence of SEQ ID No. 170, or variants thereof each having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
The CD84 binding domain may be an scFv. The scFv may comprise a heavy chain variable region (VH) and a light chain variable region (VL) of the antibody linked using short linker peptides, e.g., about 10 to 25 amino acids. The orientation of the scFv (from N-terminus to C-terminus) may be VH-VL or VL-VH. In some embodiments, the orientation of the scFv (from N-terminus to C-terminus) is VH-VL. In some embodiments, the orientation of the scFv (from N-terminus to C-terminus) is VL-VH.
Exemplary linker sequences for linking the VH domain and the VL domain include:
GGGGSGGGGSGGGGSGGGGAS(SEQ ID NO:67)
GGGGSGGGGSGGGGS(SEQ ID NO:110)
GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:111)
The antigen binding domain may comprise or consist of: SEQ ID NO: 61. 63, 112-125 or 181-184, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
Suitably, the variant binds CD84 at least as well as the corresponding antigen binding domain as shown in SEQ ID NO. 61, 63, 112-125 or 181-184. For example, a variant may specifically bind to CD84 with a binding affinity that is at least equivalent to the binding affinity between the corresponding antigen binding domain as set forth in SEQ ID NO. 61, 63, 112-125 or 181-184 and CD84.
In some embodiments, the antigen binding domain comprises or consists of: the sequence of SEQ ID NO. 61, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto and preferably retaining the ability to bind CD 84.
In some embodiments, the antigen binding domain comprises or consists of: the sequence of SEQ ID NO. 63, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto and preferably retaining the ability to bind CD 84.
In some embodiments, the antigen binding domain comprises or consists of: the sequence of SEQ ID NO. 112, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto and preferably retaining the ability to bind CD 84.
In some embodiments, the antigen binding domain comprises or consists of: the sequence of SEQ ID NO. 113, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto and preferably retaining the ability to bind CD 84.
In some embodiments, the antigen binding domain comprises or consists of: the sequence of SEQ ID NO. 114, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto and preferably retaining the ability to bind CD 84.
In some embodiments, the antigen binding domain comprises or consists of: the sequence of SEQ ID NO. 115, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto and preferably retaining the ability to bind CD 84.
In some embodiments, the antigen binding domain comprises or consists of: the sequence of SEQ ID NO. 116, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto and preferably retaining the ability to bind CD 84.
In some embodiments, the antigen binding domain comprises or consists of: the sequence of SEQ ID NO. 117, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto and preferably retaining the ability to bind CD 84.
In some embodiments, the antigen binding domain comprises or consists of: 118, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto and preferably retaining the ability to bind CD 84.
In some embodiments, the antigen binding domain comprises or consists of: the sequence of SEQ ID NO. 119, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto and preferably retaining the ability to bind CD 84.
In some embodiments, the antigen binding domain comprises or consists of: the sequence of SEQ ID NO. 120, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto and preferably retaining the ability to bind CD 84.
In some embodiments, the antigen binding domain comprises or consists of: the sequence of SEQ ID NO. 121, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto and preferably retaining the ability to bind CD 84.
In some embodiments, the antigen binding domain comprises or consists of: the sequence of SEQ ID NO. 122, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto and preferably retaining the ability to bind CD 84.
In some embodiments, the antigen binding domain comprises or consists of: the sequence of SEQ ID NO. 123, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto and preferably retaining the ability to bind CD 84.
In some embodiments, the antigen binding domain comprises or consists of: the sequence of SEQ ID NO. 124, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto and preferably retaining the ability to bind CD 84.
In some embodiments, the antigen binding domain comprises or consists of: the sequence of SEQ ID NO. 125, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto and preferably retaining the ability to bind CD 84.
In some embodiments, the antigen binding domain comprises or consists of: the sequence of SEQ ID NO:181, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto and preferably retaining the ability to bind CD 84.
In some embodiments, the antigen binding domain comprises or consists of: the sequence of SEQ ID NO. 182, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto and preferably retaining the ability to bind CD 84.
In some embodiments, the antigen binding domain comprises or consists of: the sequence of SEQ ID NO 183, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto and preferably retaining the ability to bind CD 84.
In some embodiments, the antigen binding domain comprises or consists of: the sequence of SEQ ID NO. 184, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto and preferably retaining the ability to bind CD 84.
The antigen binding domain may be comprised in a Chimeric Antigen Receptor (CAR).
Chimeric Antigen Receptor (CAR)
As used herein, a "chimeric antigen receptor" (CAR or CAR) refers to an engineered receptor that can confer antigen specificity onto a cell (e.g., a T cell, such as a naive T cell, a central memory T cell, an effector memory T cell, or a combination thereof). CARs are also known as artificial T cell receptors, chimeric T cell receptors or chimeric immune receptors. The CARs may also confer antigen specificity onto other immune cells such as NK cells (e.g., from umbilical Cord Blood (CB), induced Pluripotent Stem Cells (iPSC), bone Marrow (BM), human embryonic stem cells (hESC), cell lines (e.g., NK92 or YT) or Peripheral Blood (PB) (Mehta et al (2018) front. Immunol.9:283; liu et al (2020) N.Engl. J. Med. 382:545-553). CARs for use with NK cells may have other transmembrane domains (such as NKG2D or DAP 12) and other co-stimulatory domains (such as NKG2D or 2B 4), and they may incorporate IL-2 or IL-15 genes in the CAR construct to provide cytokine support for the CAR-NK cells constantly.
For example, the CAR may comprise an antigen binding domain, a transmembrane domain, and an intracellular signaling domain (intracellular domain).
In some embodiments, the CAR comprises a CD84 binding domain, a transmembrane domain, and an intracellular signaling domain. The CAR may comprise one or more co-stimulatory domains.
The antigen binding domain of the CAR (e.g., CD84 binding domain) can be an antigen binding domain as disclosed herein.
In another aspect, the invention provides a Chimeric Antigen Receptor (CAR) comprising or consisting of: 172-180, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
Suitably, the variant may function at least as well as the corresponding CAR as shown in SEQ ID NOS 172-180. For example, the variant can specifically bind to CD84 with a binding affinity that is at least equivalent to the binding affinity between the corresponding CAR and CD84 as shown in SEQ ID NOS 172-180.
In another aspect, the invention provides a Chimeric Antigen Receptor (CAR) comprising or consisting of: the sequence of SEQ ID NO. 172, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
In another aspect, the invention provides a Chimeric Antigen Receptor (CAR) comprising or consisting of: the sequence of SEQ ID NO 173, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
In another aspect, the invention provides a Chimeric Antigen Receptor (CAR) comprising or consisting of: the sequence of SEQ ID NO. 174, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
In another aspect, the invention provides a Chimeric Antigen Receptor (CAR) comprising or consisting of: the sequence of SEQ ID NO. 175, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
In another aspect, the invention provides a Chimeric Antigen Receptor (CAR) comprising or consisting of: the sequence of SEQ ID NO. 176, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
In another aspect, the invention provides a Chimeric Antigen Receptor (CAR) comprising or consisting of: the sequence of SEQ ID NO. 177, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
In another aspect, the invention provides a Chimeric Antigen Receptor (CAR) comprising or consisting of: the sequence of SEQ ID NO. 178, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
In another aspect, the invention provides a Chimeric Antigen Receptor (CAR) comprising or consisting of: the sequence of SEQ ID NO. 179, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
In another aspect, the invention provides a Chimeric Antigen Receptor (CAR) comprising or consisting of: the sequence of SEQ ID NO. 180, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
Transmembrane domain
The CAR may comprise a transmembrane domain.
The transmembrane domain may comprise a transmembrane sequence from any protein having a transmembrane domain, including any of the type I, type II or type III transmembrane proteins. The transmembrane domain of the CAR may also comprise an artificial hydrophobic sequence. The transmembrane domain of the CAR may be selected so that dimerization does not occur.
Examples of Transmembrane (TM) regions for CAR constructs are: a) CD28 (TM) region (Puleo et al, mol Ther,2005, nov;12 (5) 933-41; brentjens et al, CCR,2007, sep15;13 (18 Pt 1) 5426-35; casucci et al, blood,2013, nov 14;122 3461-72); b) OX40TM region (Puleo et al, mol Ther,2005, nov;12 (5) 933-41); c) The 4-1BB TM region (Brentjens et al, CCR,2007, sep15;13 (18 Pt 1): 5426-35); d) CD3- ζ TM region (Pulnet al, mol Ther,2005, nov;12 (5) 933-41; di Stasi et al Blood 2009, jun 18;113 6392-402); e) CD8a TM region (Maher et al, nat Biotechnol,2002, jan;20 (1) 70-5; imai C et al, leukemia,2004, apr;18 (4) 676-84; brentjens et al, CCR,2007, sep15;13 (18 Pt 1) 5426-35; milone et al, mol ter, 2009, aug;17 1453-64.); f) DAP12TM region (Muller, N.et al J.ImmunotherS.2015, june 01;38,197) of the formula (i); g) 2B4TM region (Altvater, B. Et al clin. Cancer Res,2009, july 22; (15) 4857-4866), and h) the NKG2D TM region (Li, Y et al CELL STEM CELL,2018, aug 2; (23):181-192).
Additional transmembrane domains will be apparent to those skilled in the art.
Examples of amino acid sequences of the CD8a transmembrane domain are:
IYIWAPLAGTCGVLLLSLVITLYC
(SEQ ID NO:127)
examples of amino acid sequences of the CD28 transmembrane domain are:
FWVLVVVGGVLACYSLLVTVAFIIFWV
(SEQ ID NO:128)
In some embodiments, the transmembrane domain comprises or consists of: 127 or 128, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
Signal peptides
The CAR may comprise a signal peptide such that when the CAR is expressed in a cell, the protein is directed to the endoplasmic reticulum and subsequently to the cell surface.
The signal peptide may be recognized and cleaved by a signal peptidase during or after transport to produce the mature protein.
In some embodiments, the signal peptide is a CD8a signal peptide.
Spacer(s)
The CAR may comprise a spacer that links the antigen binding domain to the transmembrane domain.
The spacer sequence may, for example, comprise an IgG1Fc region, an IgG1 hinge, or a human or mouse CD8 stem (stalk).
In some embodiments, the CAR comprises a CD8a stem.
Intracellular signaling domains
The intracellular domain may provide signaling in the CAR.
The intracellular signaling domain may comprise one or more immune receptor tyrosine activation motifs (ITAMs), which are conserved sequences of four amino acids that are repeated twice in the cytoplasmic tail of certain cell surface proteins of the immune system. The motif contains tyrosine, which is separated from leucine or isoleucine by any two other amino acids (YxxL/I). Tyrosine residues of these motifs can be phosphorylated upon interaction of the receptor molecule with its ligand and can form binding sites for other proteins involved in the signaling pathway.
The intracellular signaling domain may comprise or consist of the CD 3-zeta intracellular domains of three ITAMs. The CD 3-zeta intracellular domain can transmit an activation signal to T cells upon antigen binding.
The CAR may comprise one or more co-stimulatory domains. For example, 4-1BB (also known as CD 137) may be used with CD3- ζ, or CD28, OX40, and/or ICOS may be used with CD3- ζ to deliver proliferation/survival signaling. In addition, NKG2D, 2B4 (also known as CD 244), DAP12, and/or DAP10 can be used with CD3- ζ to deliver proliferation/survival signaling.
In some embodiments, the CAR comprises a CD 3-zeta signaling domain and lacks any co-stimulatory domain. In some embodiments, the CAR comprises a CD 3-zeta signaling domain and one or more co-stimulatory domains.
In some embodiments, the CAR comprises one or more co-stimulatory domains selected from the group consisting of a 4-1BB co-stimulatory domain, a CD28 co-stimulatory domain, and an OX40 co-stimulatory domain.
In a preferred embodiment, the CAR comprises a 4-1BB co-stimulatory domain. In some embodiments, the CAR comprises a CD28 co-stimulatory domain. In some embodiments, the CAR comprises an OX40 co-stimulatory domain.
In a preferred embodiment, the CAR comprises a 4-1BB co-stimulatory domain and a CD 3-zeta signaling domain.
An example amino acid sequence of the CD 3-zeta signaling domain is:
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
(SEQ ID NO:129)
In some embodiments, the signaling domain comprises or consists of: the sequence of SEQ ID NO. 129, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
An example amino acid sequence of the 4-1BB co-stimulatory domain is:
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
(SEQ ID NO:130)
An example amino acid sequence of the CD28 co-stimulatory domain is:
RSKRSRLLHSDYMNMTPRRPGPTRKHQYPYAPPRDFAAYRS
(SEQ ID NO:171)
In some embodiments, the co-stimulatory domain comprises or consists of: 130 or 171, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.
Polynucleotide
The polynucleotide of the invention may comprise DNA or RNA, preferably DNA. They may be single-stranded or double-stranded. Preferably, the polynucleotide is an isolated polynucleotide. Those of skill in the art will appreciate that many different polynucleotides may encode the same polypeptide due to the degeneracy of the genetic code. In addition, it will be appreciated that the skilled artisan can make nucleotide substitutions using conventional techniques that do not affect the polypeptide sequence encoded by the polynucleotides of the invention, which reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed.
The polynucleotides may be modified by any method available in the art. Such modifications may be made to enhance the in vivo activity or longevity of the polynucleotides of the invention.
Polynucleotides such as DNA polynucleotides may be produced by recombinant, synthetic, or any means known to those of skill in the art. They can also be cloned by standard techniques.
Longer polynucleotides will typically be produced using recombinant means, for example using Polymerase Chain Reaction (PCR) cloning techniques. This would involve preparing pairs of primers (e.g., about 15 to 30 nucleotides) flanking the target sequence of the desired clone, contacting the primers with mRNA or cDNA obtained from animal or human cells, performing PCR under conditions that cause amplification of the desired region, isolating the amplified fragments (e.g., by purifying the reaction mixture with agarose gel), and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA may be cloned into a suitable vector.
The polynucleotide may comprise a promoter and/or enhancer operably linked to one or more nucleotide sequences encoding an antigen binding domain, antibody or CAR of the invention. As used herein, the term "operably linked" may mean that the two components are joined together in a manner such that the two are capable of functioning substantially unimpeded. For example, a promoter and/or enhancer may promote and/or enhance expression of an antigen binding domain, antibody, or CAR.
In some embodiments, the promoter is an EF 1a promoter.
Carrier body
In some embodiments, the polynucleotide is a vector.
Preferably, the vector is a viral vector, such as a retroviral vector, a lentiviral vector, an adeno-associated viral (AAV) vector, or an adenoviral vector. In some embodiments, the polynucleotide is a viral genome.
In another aspect, the invention provides a viral vector comprising a polynucleotide of the invention.
In some embodiments, the viral vector is in the form of a viral vector particle.
The carrier is a tool that allows or facilitates the transfer of entities from one environment to another. According to the present invention, and by way of example, some vectors for recombinant nucleic acid technology allow transfer of entities, such as nucleic acid fragments (e.g., heterologous DNA fragments, such as heterologous cDNA fragments), into target cells. The carrier may serve the following purposes: maintaining the heterologous nucleic acid (DNA or RNA) within the cell, promoting replication of the vector comprising the nucleic acid fragment, and/or promoting expression of the protein encoded by the nucleic acid fragment.
Vectors comprising the polynucleotides used in the invention may be introduced into cells using a variety of techniques known in the art, such as transfection, transduction, and transformation.
Transfection may refer to the general process of incorporating nucleic acids into cells and includes the process of delivering polynucleotides to cells using non-viral vectors. Transduction may refer to the process of incorporating nucleic acids into cells using viral vectors.
Retrovirus and lentiviral vectors
The retroviral vector may be derived or derivable from any suitable retrovirus. A number of different retroviruses have been identified. Examples include Murine Leukemia Virus (MLV), human T cell leukemia virus (HTLV), mouse Mammary Tumor Virus (MMTV), rous Sarcoma Virus (RSV), bowman sarcoma virus (FuSV), moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBRMSV), moloney murine osteosarcoma virus (Mo-MSV), abelsen murine leukemia virus (A-MLV), avian myeloblastosis virus-29 (MC 29), and Avian Erythroblastosis Virus (AEV). A detailed list of Retroviruses can be found in Coffin, J.M. et al (1997) Retroviruses, cold Spring Harbour Laboratory Press, 758-63.
Retroviruses can be broadly divided into two categories, "simple" and "complex". Retroviruses can be divided even further into seven groups. Five of which represent retroviruses with oncogenic potential. The remaining two groups are lentiviruses and foamy viruses.
The basic structure of retroviral and lentiviral genomes has many common features, such as the 5'LTR and the 3' LTR. Located between or within these are packaging signals that enable packaging of the genome, primer binding sites, integration sites that enable integration of the genome into the host cell genome, and gag, pol and env genes encoding packaging components, which are polypeptides required for viral particle assembly. Lentiviruses have additional features such as rev and RRE sequences in HIV, which enable efficient export of RNA transcripts of the integrated provirus from the nucleus into the cytoplasm of the infected target cells.
In provirus, these genes are flanked at both ends by regions called Long Terminal Repeats (LTRs). The LTR is responsible for proviral integration and transcription. The LTR also acts as an enhancer-promoter sequence and can control the expression of viral genes.
LTRs themselves are identical sequences, which can be divided into three elements: u3, R and U5. U3 is derived from a sequence unique to the 3' end of RNA. R is derived from sequences repeated at both ends of the RNA. U5 is derived from a sequence unique to the 5' end of RNA. The sizes of these three elements can vary widely from retrovirus to retrovirus.
Gag, pol and env may be absent or non-functional in the defective retroviral vector genome.
In a typical retroviral vector, at least a portion of one or more protein coding regions necessary for replication may be removed from the virus. This makes the viral vector replication defective.
Lentiviral vectors are part of a larger group of retroviral vectors. A detailed lentiviral list can be found in Coffin, J.M. et al (1997) Retroviruses, cold Spring Harbour Laboratory Press, 758-63. Briefly, lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to, human Immunodeficiency Virus (HIV), pathogens of human acquired immunodeficiency syndrome (AIDS) (causative agent); and Simian Immunodeficiency Virus (SIV). Examples of non-primate lentiviruses include the prototype "chronic virus" visna/meyd virus (VMV), and the related caprine arthritis-encephalitis virus (CAEV), equine Infectious Anemia Virus (EIAV), and the recently described Feline Immunodeficiency Virus (FIV) and Bovine Immunodeficiency Virus (BIV).
The lentivirus family differs from retroviruses in that lentiviruses have the ability to infect dividing cells and non-dividing cells (Lewis, P et al (1992) EMBO J.11:3053-8; lewis, P.F. et al (1994) J.Virol.68:510-6). In contrast, other retroviruses, such as MLV, are unable to infect non-dividing or slowly dividing cells, such as cells that make up, for example, muscle, brain, lung, and liver tissue.
As used herein, a lentiviral vector is a vector comprising at least one component part derivable from a lentivirus. Preferably, the component part involves a biological mechanism by which the vector infects cells, expresses genes, or replicates.
The lentiviral vector may be a "primate" vector. Lentiviral vectors may be "non-primate" vectors (i.e., derived from viruses that do not primarily infect primates, particularly humans). An example of a non-primate lentivirus can be any member of the lentiviraceae family that does not naturally infect primates.
As an example of lentiviral-based vectors, HIV-1 and HIV-2 based vectors are described below.
HIV-1 vectors contain cis-acting elements that are also found in simple retroviruses. It has been shown that sequences extending into the gag open reading frame are important for the packaging of HIV-1. Thus, HIV-1 vectors typically contain relevant portions of gag in which the translation initiation codon has been mutated. In addition, most HIV-1 vectors also contain a portion of the env gene containing RRE. Rev binds to RRE, which allows full-length or single-spliced mRNA to be transported from the nucleus to the cytoplasm. In the absence of Rev and/or RRE, full length HIV-1RNA accumulates in the nucleus. Alternatively, constitutive transport elements from certain simple retroviruses (such as Mason-Pfizer monkey virus) can be used to alleviate the need for Rev and RRE. Efficient transcription from the HIV-1LTR promoter requires the viral protein Tat.
Most HIV-2 based vectors are very similar in structure to HIV-1 vectors. Similar to HIV-1 based vectors, HIV-2 vectors also require RRE to efficiently transport full-length or singly spliced viral RNA.
Preferably, the viral vectors used in the present invention have a minimal viral genome.
By "minimal viral genome", it is understood that viral vectors have been manipulated to remove non-essential elements and retain essential elements, thereby providing the functions required to infect, transduce and deliver a nucleotide sequence of interest to a target host cell. Further details of this strategy can be found in WO 1998/017815.
Preferably, the plasmid vector used to produce the viral genome within the host cell/packaging cell will have sufficient lentiviral genetic information to allow packaging of the RNA genome into viral particles capable of infecting the target cell but incapable of independent replication to produce infectious viral particles within the final target cell in the presence of packaging components. Preferably, the vector lacks a functional gag-pol and/or env gene and/or other genes necessary for replication.
However, the plasmid vector used to produce the viral genome within the host cell/packaging cell will also comprise transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in the host cell/packaging cell. These regulatory sequences may be native sequences associated with the transcribed viral sequence (i.e., the 5' u3 region), or they may be heterologous promoters, such as another viral promoter (e.g., the CMV promoter).
The vector may be a self-inactivating (SIN) vector in which viral enhancer and promoter sequences have been deleted. SIN vectors can be generated and transformed into non-dividing cells in vivo, with efficacy similar to wild-type vectors. Transcriptional inactivation of the Long Terminal Repeat (LTR) in SIN provirus should prevent mobilization of the virus by replication activity. This also allows for regulated expression of genes from internal promoters by eliminating any cis-action of the LTR.
The vector may be integration defective. Integration-defective lentiviral vectors (IDLV) can be generated, for example, by: the vector is packaged with a catalytically inactive integrase, such as HIV integrase carrying a D64V mutation in the catalytic site; naldini, L. Et al (1996) Science 272:263-7; naldini, L. Et al (1996) Proc. Natl. Acad. Sci. USA 93:11382-8; leavitt, A.D. Et al (1996) J. Virol. 70:721-8), or by modification or deletion of the necessary att sequence from the vector LTR (NIGHTINGALE, S.J. Et al (2006) mol. Ther. 13:1121-32), or by a combination of the above.
Cells
In another aspect, the invention provides a cell comprising a polynucleotide, vector, antigen binding domain, or CAR of the invention.
In some embodiments, the cell is a T cell, lymphocyte, or stem cell, such as a hematopoietic stem cell, umbilical cord blood stem Cell (CB), or Induced Pluripotent Stem Cell (iPSC).
For example, the cells may be selected from the group consisting of: CD4 cells, CD8 cells, th0 cells, tc0 cells, th1 cells, tc1 cells, th2 cells, tc2 cells, th17 cells, th22 cells, γ/δ T cells, natural Killer (NK) cells, natural Killer T (NKT) cells, double negative T cells, naive T cells, memory stem T cells, central memory T cells, effector T cells, cytokine-induced killer (CIK) cells, hematopoietic stem cells, and induced pluripotent stem cells (ipscs).
In some embodiments, the cell is a T cell or NK cell, preferably a T cell. In some embodiments, the T cell is an autologous T cell or an allogeneic T cell.
The cells may have been isolated from the subject.
The cells of the invention may be provided for adoptive cell transfer. As used herein, the term "adoptive cell transfer" refers to the administration of a population of cells to a patient. Typically, the cells are T cells isolated from a subject and then genetically modified and cultured in vitro prior to being administered to a patient.
Adoptive cell transfer may be allogeneic or autologous.
By "autologous cell transfer", it is understood that the starting cell population (which is subsequently transduced with a polynucleotide or vector according to the invention) is obtained from the same subject as the subject to which the transduced cell population was administered. Autograft is advantageous because it avoids the problems associated with immune incompatibilities and is available to the subject whether or not genetically matched donors are available.
By "allogeneic cell transfer", it is understood that the starting cell population (which is subsequently transduced with a polynucleotide or vector according to the invention) is obtained from a subject different from the subject to whom the transduced cell population is administered. Preferably, the donor will be genetically matched to the subject to which the cells are administered to minimize the risk of immune incompatibility. Or the donor may be mismatched or unrelated to the patient.
Method of treatment
In another aspect, the invention provides an antigen binding domain, antibody, CAR, polynucleotide, vector, cell or pharmaceutical composition of the invention for use in therapy.
In some embodiments, the therapy is cancer therapy.
In a preferred embodiment, the cancer is a hematological malignancy.
In a preferred embodiment, the cancer cell expresses CD84, e.g., the hematological malignancy may be a hematological malignancy that expresses CD 84.
In some embodiments, the cancer is selected from the group consisting of: chronic Lymphocytic Leukemia (CLL), B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), burkitt's lymphoma, follicular lymphoma, mantle cell lymphoma, B-cell acute lymphoblastic leukemia (B-ALL), acute Myeloid Leukemia (AML), myelodysplastic syndrome, T-cell acute lymphoblastic leukemia/lymphoma (T-ALL), chronic myeloproliferative syndrome, chronic Myeloid Leukemia (CML), chronic myelomonocytic leukemia, dendritic cell neoplasm, and histiocytosarcoma.
In some embodiments, the cancer is selected from the group consisting of: chronic Lymphocytic Leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), burkitt's lymphoma, follicular lymphoma, mantle cell lymphoma, B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia/lymphoma (T-ALL), acute Myeloid Leukemia (AML), and histiocyte sarcoma.
In a preferred embodiment, the cancer is selected from the group consisting of CLL, B-cell lymphoma, B-ALL, T-ALL and AML.
In a preferred embodiment, the cancer is selected from the group consisting of B-cell lymphoma, B-ALL, T-ALL, and AML.
In another aspect, the invention provides an antigen binding domain, antibody, CAR, polynucleotide, vector, cell or pharmaceutical composition of the invention for use in the treatment of AML.
In another aspect, the invention provides an antigen binding domain, antibody, CAR, polynucleotide, vector, cell or pharmaceutical composition of the invention for use in the treatment of T-ALL.
In another aspect, the invention provides an antigen binding domain, antibody, CAR, polynucleotide, vector, cell or pharmaceutical composition of the invention for use in the treatment of B-cell lymphoma.
In another aspect, the invention provides an antigen binding domain, antibody, CAR, polynucleotide, vector, cell or pharmaceutical composition of the invention for use in the treatment of burkitt's lymphoma.
In some embodiments, the cancer is not a T cell lymphoma. In some embodiments, the cancer is not mature T cell lymphoma. In some embodiments, the cancer is not CLL. In some embodiments, the cancer is not a solid tumor.
Preferably, the mammal, in particular the human, is treated. Both human and veterinary treatments are within the scope of the present invention.
Pharmaceutical composition and injection solution
Although the agents used in the present invention may be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human therapy.
The medicament of the invention, e.g. a cell or carrier particle, may be formulated as a pharmaceutical composition. In addition to the drug, these compositions may also contain pharmaceutically acceptable carriers, diluents, excipients, buffers, stabilizers or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can be determined by one skilled in the art based on the route of administration (e.g., intravenous or intra-arterial).
The pharmaceutical composition is typically in the form of a liquid. Liquid pharmaceutical compositions typically include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral or synthetic oils. May include physiological saline solution, magnesium chloride, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. In some cases, surfactants such as pluronic acid (PF 68) 0.001% may be used. In some cases, serum albumin may be used in the composition.
For injectable formulations, the active ingredient may be in the form of an aqueous solution which is pyrogen-free and has a suitable pH, isotonicity and stability. Those skilled in the art will be able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, ringer's injection or lactated ringer's injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included as desired.
For delayed release, the drug may be included in a pharmaceutical composition formulated for slow release according to methods known in the art, such as in microcapsules formed of biocompatible polymers or in a liposome carrier system.
The treatment of the cell therapy product is preferably performed according to the FACT-JACIE international standard of cell therapy.
Application of
In some embodiments, the antigen binding domain, antibody, CAR, polynucleotide, vector, cell, or pharmaceutical composition of the invention is administered systemically to a subject.
In some embodiments, the antigen binding domain, antibody, CAR, polynucleotide, vector, cell, or pharmaceutical composition of the invention is topically administered to a subject.
As used herein, the term "systemic delivery" or "systemic administration" refers to administration of an agent of the invention into the circulatory system, for example, to achieve a broad distribution of the agent. In contrast, external or topical administration limits the delivery of the agent in a localized area.
In some embodiments, the antigen binding domain, antibody, CAR, polynucleotide, vector, cell, or pharmaceutical composition of the invention is administered intravascularly, intravenously, or intraarterially.
In a preferred embodiment, the antigen binding domain, antibody, CAR, polynucleotide, vector, cell or pharmaceutical composition of the invention is administered intravenously.
Dosage of
One skilled in the art can readily determine the appropriate dosage of the agent of the invention to administer to a subject. In general, the physician will determine the actual dosage which will be most suitable for an individual patient and this will depend on a number of factors, including the activity of the particular compound used, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. Of course, there may be individual cases where higher or lower dosage ranges are reasonable (merited), and such cases are within the scope of the present invention.
A subject
As used herein, the term "subject" refers to a human or non-human animal.
Examples of non-human animals include vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), dogs, rodents (e.g., mice, rats, or guinea pigs), pigs, and cats. The non-human animal may be a companion animal.
Preferably, the subject is a human.
Variants, derivatives, analogs, homologs and fragments
In addition to the specific proteins and nucleotides mentioned herein, variants, derivatives, analogs, homologs and fragments thereof are also encompassed by the present invention.
In the context of the present invention, a "variant" of any given sequence refers to a sequence in which a particular sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a way that the polypeptide or polynucleotide in question retains at least one of its endogenous functions. Variant sequences may be obtained by addition, deletion, substitution, modification, replacement and/or alteration of at least one residue present in a naturally occurring polypeptide or polynucleotide.
As used herein, the term "derivative" in connection with a protein or polypeptide of the invention includes any substitution, variation, modification, substitution, deletion and/or addition of one (or more) amino acid residues from or to a sequence, provided that the resulting protein or polypeptide retains at least one of its endogenous functions.
As used herein, the term "analog" in relation to a polypeptide or polynucleotide includes any mimetic, i.e., chemical compound that has at least one endogenous function of the polypeptide or polynucleotide to which it mimics.
Typically, amino acid substitutions (e.g., from 1, 2, or 3 to 10 or 20 substitutions) can be made, provided that the modified sequence retains the desired activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogs.
Proteins for use in the present invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein. Deliberate amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids having uncharged polar head groups with similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine.
Conservative substitutions may be made, for example according to the following table. Amino acids in the same lattice in the second column, and preferably in the same row in the third column, may be substituted for each other:
As used herein, the term "homolog" means an entity that has some homology to a wild-type amino acid sequence or a wild-type nucleotide sequence. The term "homology" may be equated with "identity".
In this context, homologous sequences are considered to comprise amino acid sequences which are at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95%, 96% or 97% or 98% or 99% identical, to the subject sequence. Typically, the homologue will comprise the same active site or the like as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues with similar chemical properties/functions), in the case of the present invention it is preferred that homology is expressed in terms of sequence identity.
In this context, homologous sequences are considered to comprise nucleotide sequences which are at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95%, 96% or 97% or 98% or 99% identical, to the subject sequence. Although homology can also be considered in terms of similarity, in the case of the present invention, it is preferable to express homology in terms of sequence identity.
Preferably, reference to a sequence having a percent identity to any one of the SEQ ID NOs detailed herein refers to a sequence having said percent identity over the entire length of the SEQ ID NOs referred to.
Homology comparisons may be made by the naked eye or, more commonly, by available sequence comparison procedures. These commercially available computer programs can calculate percent homology or identity between two or more sequences.
The percent homology can be calculated over consecutive sequences, i.e., one sequence is aligned with another and each amino acid or nucleotide in one sequence is directly compared to the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is referred to as a "vacancy free" alignment. Typically, such vacancy free alignments are performed only on relatively short numbers of residues.
Although this is a very simple and consistent approach, it does not contemplate that, for example, an insertion or deletion in an amino acid or nucleotide sequence in a pair of otherwise identical sequences may result in subsequent residues or codons being unaligned, thus potentially resulting in a substantial reduction in percent homology when a global alignment is performed. Thus, most sequence comparison methods are designed to produce optimal alignments, taking into account possible insertions and deletions without unduly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment in an attempt to maximize local homology.
However, these more complex methods assign a "gap penalty" to each gap that exists in an alignment, so that for the same number of identical amino acids or nucleotides, a sequence alignment with as few gaps as possible (reflecting a higher correlation between two compared sequences) will achieve a higher score than a sequence alignment with many gaps. An "affine gap cost" is typically used that charges a relatively high cost to the existence of a gap and a small penalty to each subsequent residue in the gap. This is the most commonly used vacancy scoring system. High gap penalties will of course result in an optimal alignment with fewer gaps. Most alignment programs allow for modification of the gap penalty. But when using such software for sequence comparison, it is preferable to use default values. For example, when using the GCG Wisconsin Bestfit packets, the default gap penalty for an amino acid sequence is one gap-12 and each extension-4.
Thus, calculating the maximum percent homology first requires generating an optimal alignment, taking into account gap penalties. Suitable computer programs for this analogy are the GCG Wisconsin Bestfit package (University of Wisconsin, USA; devereux et al (1984) Nucleic ACIDS RESEARCH 12:387. Examples of other software that may perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al (1999) ibid-Ch.18), FASTA (Atschul et al (1990) J.mol. Biol. 403-410) and GENEWORKS comparison tool kits, BLAST and FASTA are both available for offline and online searches (see Ausubel et al (1999) supra, pages 7-58 to 7-60.) however, for some applications it is preferred to use the GCG Bestfit program. Another tool, BLAST 2Sequences, are also available for comparison of protein and nucleotide Sequences (FEMS Microbiol. Lett. (1999) 174: 50;FEMS Microbiol.Lett. (1999) 177-8).
Although the final percent homology can be measured in terms of identity, the alignment process itself is typically not based on an all or nothing pairing comparison. Instead, each pair comparison is assigned a score based on chemical similarity or evolutionary distance, typically using a scaled similarity score matrix. A common example of such a matrix is the BLOSUM62 matrix (default matrix of the BLAST suite of programs). The GCG Wisconsin program typically uses common default values or custom symbol comparison tables (if provided) (see user manual for details). For some applications it is preferred to use a common default value for the GCG package, or in the case of other software, a default matrix, such as BLOSUM62.
Once the software has produced the optimal alignment, the percent homology, preferably percent sequence identity, can be calculated. Software typically runs this as part of the sequence comparison and generates a numerical result.
A "fragment" is also a variant, and the term generally refers to a selected region of a polypeptide or polynucleotide that is of interest functionally or in, for example, an assay. Thus, a "fragment" refers to an amino acid or nucleic acid sequence that is part of a full-length polypeptide or polynucleotide.
Such variants can be made using standard recombinant DNA techniques (such as site-directed mutagenesis). Where insertion is desired, synthetic DNA encoding the insert may be prepared, along with 5 'and 3' flanking regions corresponding to naturally-occurring sequences flanking the insertion site. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cleaved with the appropriate enzymes and the synthetic DNA ligated into the nicks. The DNA is then expressed according to the invention to produce the encoded protein. These methods are merely illustrative of the many standard techniques known in the art for manipulating DNA sequences and other known techniques that may also be used.
Codon optimization
Polynucleotides for use in the present invention may be codon optimized. Codon optimisation has been described previously in WO 1999/41397 and WO 2001/79518. Different cells differ in the choice of their particular codons. This codon bias corresponds to the bias in the relative abundance of a particular tRNA in a cell type. Expression can be increased by altering codons in the sequence to match the relative abundance of the corresponding tRNA. For the same reason, expression can be reduced by deliberately selecting codons for which the corresponding tRNA is known to be rare in a particular cell type. Thus, an additional degree of translational control is available. Codon usage tables are known in the art for mammalian cells and for a variety of other organisms.
Those skilled in the art will appreciate that they can combine all of the features of the invention disclosed herein without departing from the scope of the invention as disclosed.
Preferred features and embodiments of the present invention will now be described by way of non-limiting examples.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the ability of a person of ordinary skill in the art. Such techniques are explained in the literature. See, e.g., sambrook, j., fritsch, e.f., and Maniatis,T.(1989)Molecular Cloning:ALaboratory Manual,2nd Edition,Cold Spring Harbor Laboratory Press;Ausubel,F.M. et al (1995 and periodic supplements) Current Protocols in Molecular Biology, ch.9,13, and 16, john Wiley and Sons; each of these general texts are incorporated herein by reference, for example, roe, b., crabtree, j. And Kahn, a. (1996) DNAIsolation and Sequencing: ESSENTIAL TECHNIQUES, john Wiley and Sons;Polak,J.M.and McGee,J.O'D.(1990)In Situ Hybridization:Principles and Practice,Oxford University Press;Gait,M.J.(1984)Oligonucleotide Synthesis:APractical Approach,IRL Press;, and liley, d.m., and Dahlberg,J.E.(1992)Methods in Enzymology:DNAStructures Part A:Synthesis and Physical Analysis of DNA,Academic Press..
Examples
Example 1: expression of CD84 in hematological malignancies
CD84 has been observed to be overexpressed in a range of malignant hematological diseases. Using an interactive web server GEPIA for analysis of RNA sequencing expression data from 9,736 tumors and 8,587 normal samples of TCGA and GTEx project (http:// gepia. Cancer-pku. Cn/index. Html; tang et al GEPIA:a web server for cancer and normal gene expression profiling and interactive analyses.Nucleic Acids Res.2017;10.1093/nar/gkx247), has found:
CD84 expression in 47 lymphoid lineage neoplasms diffuse large B cell lymphomas (shown as DLBC in fig. 1) was 4.2-fold higher than CD84 expression in 337 blood samples.
CD84 expression was 10.1-fold higher in 173 acute myeloid leukemia (represented as LAML in fig. 1) than in 70 bone marrow samples.
Also queried was CANCER CELL LINE Encyclopaedia (CCLE) dataset containing mRNA expression data from more than 1,100 cell lines of different types of solid and hematological tumors (https:// ports. Broadenstitute. Org/ccle). Both RNAseq and Affymetrix analysis of mRNA expression showed that CD84 was expressed at high levels in cell lines derived from burkitt's lymphoma, acute Myeloid Leukemia (AML), B cell lymphoma, chronic Myeloid Leukemia (CML), B cell acute lymphoblastic leukemia (B-ALL), and T cell acute lymphoblastic leukemia (T-ALL), but not in T cell lymphoma or solid tumors (fig. 2).
Then, we assessed the expression of CD84 on the surface of cell lines derived from different hematological malignancies. Table 1 shows the cell lines used, hematological malignancies of their origin, and qualitative assessment of CD84 expression based on the cell count analysis shown in fig. 3.
Table 1A list of cell lines derived from hematological malignancies for evaluation of surface CD84 expression and qualitative assessment of CD84 expression (acute myeloblastic leukemia cell line described herein as MOLM-13, previously designated as acute myeloblastic leukemia cell line Kasumi-1; references to Kasumi-1 have been updated accordingly).
| Cell lines | Malignant tumor of blood system | CD84 expression |
| Ramos | Burkitt's lymphoma | High height |
| Raji | Burkitt's lymphoma | High height |
| Daudi | Burkitt's lymphoma | High height |
| NALM6 | B cell acute lymphoblastic leukemia | High height |
| MOLT-4 | Acute lymphoblastic leukemia of T cells | High height |
| U937 | Histiocyte lymphoma | Low and low |
| MOLM-13 | Acute myeloblastic leukemia | Medium and medium |
| K562 | Chronic myelogenous leukemia (acute stage) | Low and low |
| THP-1 | Acute monocytic leukemia | Low and low |
CD84 expression in 9 samples from patients with CLL was analyzed by flow cytometry. Table 2 shows a qualitative assessment of CD84 expression based on the cell count analysis shown in fig. 4. CD84 expression on leukemia cells was compared to CD84 expression in lymphocytes (CD 84 Medium and medium) and monocytes (CD 84 High height). The light gray histogram represents staining with isotype matched control antibody and the dark gray histogram represents staining with specific CD84 antibody.
Table 2. Expression of CD84 in leukemia cells from 9 patients with Chronic Lymphocytic Leukemia (CLL) was assessed by flow cytometry.
Although some genomic databases indicate that CD84 is overexpressed at mRNA level in AML, we attempted to confirm whether CD84 is expressed on the surface of malignant cells from patients diagnosed with AML (n=10) (table 3).
Table 3. Expression of CD84 in leukemia cells of 10 patients with Acute Myeloid Leukemia (AML) was assessed by flow cytometry.
| Patient numbering | CD84 expression |
| P01 | Medium and medium |
| P02 | Medium and medium |
| P03 | High height |
| P04 | Medium and medium |
| P05 | Medium and medium |
| P06 | Medium and medium |
| P07 | Medium and medium |
| P08 | Medium and medium |
| P09 | Medium and medium |
| P10 | High height |
Fig. 5 shows two representative examples of flow cytometry data: samples from patient 04 and patient 10 showed medium and high CD84 expression, respectively.
Example 2: CD84 antibodies
Murine monoclonal antibodies
Two anti-CD 84 murine monoclonal antibodies (152-1D 5 and 153-4D9; engel et al B-CELL ANTIGENS section report, in Schlossman S (ed.): leucocyte Typing V.Oxford, UK, oxford University Press,1995,page 483;Palou et al Genomic characterization of CD84reveals the existence of five isoforms differing in their cytoplasmic domains.Tissue Antigens.2000;55(2):118-27))) were studied and identified.
In addition, by immunizing BALB/c mice with human CD84 protein, a number of novel anti-CD 84 mouse monoclonal antibodies were generated as described in the methods section. Table 3 summarizes the characteristics of these antibodies, as characterized in the methods section. Binding to 300.19-CD84+, raji and Ramos cells, lymphocytes and monocytes was performed using hybridoma supernatants having an equal amount/concentration of anti-CD 84 antibody.
Table 4. Characterization of anti-CD 84 murine monoclonal antibodies.
Sequences corresponding to the variable region (VL) from the light chain and sequences corresponding to the variable region (VH) from the heavy chain were determined from different hybridomas using Mouse Ig-PRIMER SET (Novagen). Mulberry sequencing of this region was performed by AbsoluteAntibody (United Kingdom). Antibody sequence analysis was performed as follows: complementarity Determining Regions (CDRs) and Framework Regions (FR) were identified using the Abysis (sequence defined by Kabat numbering scheme), IMGT and IgBLAST databases.
Table 5 shows the VH and VL sequences of these antibodies. The three CDRs in each sequence are highlighted in bold and underlined.
Table 5. VH and VL sequences of anti-CD 84 murine monoclonal antibodies.
Human scFv
Phage display screening was used to identify novel human scFv that bind to CD 84. Three different scFvs (R3-B3, R3-G7 and R3-H3) were identified; CDR and FR sequences in the variable region of the heavy chain (VH) and the variable region of the light chain (VL) are shown in table 6.
Table 6 VL and VH sequences of anti-CD 84 human scFv.
Example 3: CD84CAR engineering
Several anti-CD 84CAR constructs (CAR 84) were designed. The complete CAR84 sequence, comprising the signal peptide, scFv specific for CD84, the CD8a hinge and transmembrane regions, the co-stimulatory domain 4-1BB and the signaling domain CD3- ζ, was cloned into a third generation lentiviral vector pCCL (Dull et al AThird-Generation Lentivirus Vector with a Conditional Packaging System. J. Virol. 1998:72:8463-8471) under the control of the EF1 alpha promoter (FIG. 6).
Based on the antibodies described above, different versions of scFv domains were designed. These scfvs differ in the order of VH and VL sequences (some versions of which have only VH chains), and in the linker used between VH and VL, which use three (S3) or four (S4) motifs (Gly-Ser). The name of each CAR version reflects the design of the scFv. The sequences of the different scFv domains synthesized and cloned in the pCCL vectors are listed in table 7.
Table 7. The sequence and structure of scFv domains used in each CD84CAR were designed.
The complete CAR sequence is shown in table 8.
Table 8. Complete CAR sequence.
CD84CART Generation
Lentiviruses (LV) containing different versions of pCCL-EF1 alpha-CD 84 vectors were generated in HEK293-T cells and the number of transducing units was determined by limiting dilution. Lentiviruses were then used to transduce T cells isolated from whole blood. These CAR-T cells were then expanded for 6 to 8 days.
The following tables (tables 9-11) summarize the number of T cells obtained after three different transduction for different CART, and the percentage of these T cells expressing CD84CAR on their surface.
Table 9. Number and percentage of CAR positive T cells obtained after three independent transduction with CD84CAR expressing lentivirus based on 152-1D5 antibody.
Table 10 number and percentage of CAR positive T cells obtained after three independent transduction with CD84CAR expressing lentiviruses based on 153-4D9 antibody.
Table 11 number and percentage of CAR positive T cells obtained after three independent transduction with CD84CAR expressing lentiviruses based on R3-B3, R3-G7 and R3-H3 scFv.
The following CAR LV did not result in efficient amplification of CAR positive T cells: 152.1, 153.1, 153.2, B3.4 and B3.5. 152.1 and 153.1 CARs were not used for further experiments.
Example 4: CD84CART in vitro cytokine production
To assess the ability of each CART cell to release cytokines, supernatants of effector-target cell co-cultures were collected. CD84 High height cell line Ramos was used as target cells at a 2:1 effector to target cell ratio. After 24 hours of co-culture, the levels of IFN-gamma (FIG. 7), IL-2 (FIG. 8), granzyme-B (FIG. 9) and TNF-alpha (FIG. 10) in the supernatant were determined by enzyme-linked immunosorbent assay (ELISA). The non-transduced T cells (UTs) were used as negative controls (co-cultured with target cells). Four different independent experiments were performed.
These results show that CART cells with R3-B3scFv CAR binding domains lack cytokine release activity and thus were not used in subsequent experiments. In another aspect, CART cells with R3-H3scFv CAR binding domains release a large amount of cytokines, wherein both CART cells (H3.4 and H3.5) are the cells with the strongest pro-inflammatory profile (based on TNF- α secretion), followed by CART cells with 152-1D5 antibody CAR binding domains, which also show a high cytokine release profile. Independent of scFv design (VH-VL sequence and linker length), 152-1D5CART cells showed similar profiles. CART cells based on 153-4D9 and R3-G7 antibodies have similar profiles except 153.3CART cells, which hardly secrete cytokines, with lower cytokine release compared to R3-H3 and 152-1D5CART cells.
Example 5: anti-CD 84CART in vitro cytotoxicity
To evaluate the in vitro efficacy of CART84 cells, cytotoxicity assays were performed on each CART cell against several GFP-expressing target cell lines, which had different levels of CD84 expression and were derived from lymphoid (Ramos, NALM6 and MOLT-4 cell lines) and myeloid (K562 and MOLM-13 cell lines). After 24 hours co-culture, CART cytotoxicity was assessed by determining the percentage of viable GFP positive cells by flow cytometry. Effector cells were tested at target cell ratios of 4:1, 2:1, 1:1 and 0.5:1.
First, all CARs were tested against the CD84 High height Ramos cell line (fig. 11). Results are shown for a 2:1 effector to target ratio. R3-H3 and 152-1D5CART cells showed the highest cytotoxic activity against this cell line, whereas 153-4D9 and R3-G7CART cells showed lower cytotoxic activity. 153.3CART cells showed the lowest cytotoxic activity among all CARs based on 153-4D9 construct.
Next, all CARs were tested against K562 (myeloid cell line with low CD84 expression) (fig. 12). Results are shown for a 2:1 effector to target ratio. CARs based on 152-1D5 and 153-4D9 antibodies showed statistically significant cytotoxic activity compared to UT cells.
Based on cytotoxicity against Ramos and K562, the following CART cells were selected for further characterization: 152.3, 153.4, 153.5, G7.5 and H3.5. Cytotoxicity of these CART cells against several cell lines after 24 and 48 hours of co-culture was assessed at four effector cells to target cell ratios (4:1, 2:1, 1:1 and 0.5:1). Ramos is an invasive B-cell lymphoma cell line (fig. 13), K562 is an acute myeloid leukemia cell line with low CD84 expression (fig. 14), whereas MOLM-13 is an acute myeloid leukemia cell line with moderate CD84 expression (fig. 15). NALM-6 was a B cell acute lymphoblastic leukemia cell line (FIG. 16), and MOLT-4 was a T cell acute lymphoblastic leukemia cell line (FIG. 17).
As shown for the 2:1 ratio (fig. 11), the selected CARs showed statistically significant cytotoxic activity against Ramos cells for each effector cell: target cell ratio compared to UT cells (fig. 13). In the case of K562, the same pattern shown in fig. 12 was observed, where CARs based on the 152-1D5 and 153-4D9 antibodies showed statistically significant cytotoxic activity compared to UT cells (fig. 14).
All selected CARs showed cytotoxic activity against MOLM-13, with 152.1D5 and 153.4D9-based CARs having a higher cytotoxic effect than R3-G7 and R3-H3 antibody-based CARs (fig. 15).
CARs based on 152.1D5 and 153.4D9 antibodies showed higher cytotoxic activity against the NALM-6 cell line than CARs based on R3-G7 and R3-H3scFv (figure 16). For the MOLT-4 cell line, the 152-1D5 antibody-based CAR showed the highest statistically significant cytotoxic activity compared to UT cells, followed by 153-4D9 and R3-G7 antibody-based CAR (FIG. 17).
Example 6: CD84 expression in Peripheral Blood Mononuclear Cells (PBMC)
CD84 expression in different cell populations of PBMCs was assessed by flow cytometry and results similar to those previously described were obtained. Monocytes showed higher CD84 expression, similar to that observed in Ramos cell lines. B cells exhibit moderate CD84 expression. Both CD4 and CD8T cells have two different subsets (positive and negative) with respect to CD84 expression, with moderate CD84 expression levels on the positive population (fig. 18).
Example 7: in vitro cytotoxicity of CD84CART against PBMCs
The cytotoxic activity of CD84CART against PBMCs was assessed. For each experiment, both effector cells (i.e., CART84 cells) and target cells (PBMCs) were from the same donor. The average of three independent experiments for three different donors is shown. CART cells did not exhibit statistically significant cytotoxic activity against their own PBMCs. However, these CART cells did show statistically significant cytotoxic activity against Ramos cells in parallel experiments (fig. 19).
Example 8: methods for examples 1 to 20
Generation of murine monoclonal anti-CD 84 antibodies
BALB/c mice were immunized three to four times at 3 week intervals with 300.19 cells stably transfected with CD84 full length DNA (de la Funte MA et al CD84leukocyte ANTIGEN IS A NEW membrane of the Ig superfamily. Blood.1997;15;90 (6): 2398-405). The first intraperitoneal (i.p.) injection consisted of 300 μl of 20x106 cells in PBS, the second injection consisted of 300 μl of 20x106 cells in PBS, and the last injection consisted of 300 μl of 30x106 cells in PBS. Mice were euthanized on the third day after the last boost and spleen cells were harvested for cell fusion.
NS1 myeloma cells (European Collection of Cell Cultures, salisbury, UK) were fused with spleen cells by incubation at 37 ℃ at a ratio of 4:1 (spleen cells: NS 1), centrifugation at RT for 10 min, and 1mL of warm PEG solution was added to the cell pellet while rotating continuously. Cells were slowly resuspended in RPMI medium, centrifuged and incubated in 5% co2 at 37 ℃ in a humidified incubator.
Untransfected 300.19 cells were used as negative control and 10 days after fusion were screened by flow cytometry with 300.19-CD84 cells to analyze 50 μl hybridoma supernatant. Hybridomas that were positive for 300.19-CD84 detection and negative for 300.19 cell detection were transferred to 24-well plates, grown until confluent, and then transferred to T75 flasks. Individual hybridoma clones producing the antibody of interest are then isolated by limiting dilution. For each hybridoma, 10 monoclonal were retested by flow cytometry using 300.19-CD84 cells. The cloning protocol was repeated with one of the positive clones.
For each antibody, an anti-IgG coated plate with added antibody was used and then incubated with anti-mouse HRP antibody to determine isotype (class and subclass) by ELISA. To determine which of the two CD84 extracellular domains is recognized by the antibody, a flow cytometry analysis was performed using COS cells expressing the chimeric CD84 extracellular domains, wherein the domain 1 (D1) and domain 2 (D2) sequences were human or murine.
Identification of fully human anti-CD 84scFv
Human CD84 (checked by SDS-PAGE; FIG. 20) produced by mammalian cells was used as antigen to panning against a highly diverse library of human initial phage display LiAb SFMax with 5.37x1010 scFv variants (Proteogenix, france).
For the biopanning rounds, tubes were coated with antigen, blocked, washed and incubated with phage libraries. After washing, the phage binding agent was eluted with glycine HCl and then neutralized.
To determine the concentration of eluted phage, these were added to E.coli (E.coli) TG1 cells, which were then poured into a petri dish and cultured upside down. PFU (plaque forming unit) was calculated based on the number of plaques on the plate (i.e. dead TG 1).
To amplify the eluted phage, it was added to E.coli TG1 cells, which were then infected with helper phage and cultured. Phage precipitation with PEG/NaCl followed by re-suspension and the amplified phage were used for biopanning in the next round of ELISA analysis.
The analysis showed significant enrichment in rounds (table 12), and no additional biopanning rounds were performed to prevent a decrease in phage diversity, since round 3 output already showed excellent enrichment.
TABLE 12 concentration of eluted phages
Phages from round 3 were selected for monoclonal ELISA analysis. Individual E.coli TG1 clones were selected and cultured with helper phage. After centrifugation, the supernatant containing phage was collected.
Plates were coated with antigen or buffer, washed, blocked and washed again. Phage were then added to the plates and incubated. Plates were washed, then anti-phage horseradish peroxidase (HRP) antibody was added, then washing and incubation of TMB were performed, then HCl was added. Plates were read at 450 nm.
After sequencing the positive clones, 3 different unique sequences were identified. The 3 unique clones identified were then retested by ELISA to ensure clone specificity. All phages were tested at the same concentration. The plates are covered with antigen (Ag) or with buffer (NC). The results clearly demonstrate that 3 clones bind specifically to the CD84 antigen (table 13).
TABLE 13 ELISA of positive clones
Donor, cell line
Buffy coat of healthy donor was obtained from reference blood bank Banc de Sang i Teixits, barcelona (Spain).
Ramos, raji, NALM6, K562, MOLM-13, kasumi-6, THP-1 and U937 were purchased from AMERICAN TYPE Culture Collection (ATCC). Ramos, raji, NALM6, K562, NS1 and THP-1 cell lines were cultured in RPMI medium (ThermoFisher) supplemented with 10% fetal bovine serum (FBS, merck) and penicillin-streptomycin (Labclinics), and 300.19 cells were cultured in RPMI medium (ThermoFisher) further supplemented with 1% L-glutamine (Gibco) and 0.1% 2-beta-mercaptoethanol (Sigma). HEK293T and COS cell lines were cultured in DMEM medium (Gibco) supplemented with 10% fbs (Merck) and penicillin-streptomycin (Labclinics) and 1% l-glutamine (Gibco). All cell lines were grown at 37℃and 5% CO2.
Lentivirus production
HEK293-T cells were transfected with the applicant's transfer vector pCCL-EF 1. Alpha. -CD84 along with the packaging plasmids pRSV-Rev (Addgene, 12253), pMDLg/Prre (Addgene, 12251) and the envelope plasmid pCMV-VSV-G (Addgene, 12259) using linear polyethylenimine (PEI, molecular weight 25000,Polysciences Inc 23966-1). Lentiviral supernatants were collected after 48 hours and concentrated using LentiX-Concentrator (Clontech, takara) according to the manufacturer's protocol. Concentrated lentiviruses were stored at-80 ℃ until use.
Lentivirus titration
The number of transduction units (TU/mL) was determined by limiting dilution.
HEK293T cells were inoculated 24h prior to transduction and virus supernatants were prepared at 1:10 dilution and then added to DMEM medium (Gibco) supplemented with 8mg/mL polybrene (Sigma-Aldrich). After 48h, cells were trypsinized and labeled with AffiniPure F (ab') 2 fragment goat anti-mouse immunoglobulin G (IgG) Allophycocyanin (APC) conjugate (JacksonImmuno Research Laboratories, 115-136-072). The dilution corresponding to 2% -20% of positive cells was used to calculate the viral titer.
T cell transduction and CART expansion
T cells were isolated from whole blood during density gradient centrifugation with Ficoll-PaqueTM by rosetteepTM (rosetteep Human T CELL ENRICHMENT mixture from StemCell). T cells were cultured in X-Vivo 15Serum Free Cell Medium (Lonza) supplemented with 5% AB human serum (Sigma H4522), penicillin-streptomycin (ThermoFisher, 100 mg/mL) and 50IU/mL IL-2 (Miltenyi). Cells were then activated using beads conjugated with CD3 and CD28mAb ((Dynabeads Human T-Activator CD3/CD28Gibco, 11131D). Twenty-four hours later, they were transduced with lentivirus in the presence of 8ug/mL of polybrene (Sigma-Aldrich). 6 to 8 days of amplification was required before experiments were performed.
Flow cytometry
CAR84 uses the following detection: recombinant CD84-His protein (R & D, 1855-CD) and secondary anti-HIS TAG APC conjugated antibodies (R & D, IC 050A) and biotin SP conjugated AffiniPure F (ab ') 2 fragment goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, 115-065-072) or biotin SP conjugated AffiniPure F (ab') 2 fragment goat anti-human IgG (Jackson ImmunoResearch Laboratories, 109-065-006) and BV421 conjugated streptavidin (BD Horizon, 563259). The following mAb：CD3-APC、CD4-PE-Cy7、CD4-Alexa Fluor-488、CD19-PE、CD8-APC-H7、PD-1-PE-Cy7、TIM-3-BB515、CD69-PerCP-CyTM5.5、LAG-3-BV-605、CD62L-FITC、CCR7-PerCP-CyTM5.5、CD84-PE、CD4-PE-CyTM7、CD45Ra-aPC(Becton Dickinson)、CD84-APC and CD84-PE (BioLegend) directed against human proteins were used. Samples were run by flow cytometry BD FACSCanto II (BD Biosciences), LSR II Fortessa 4L with HTS (BD), and Attune NxT L flow cytometer (thermo fisher). For ELISA, the following mabs were used: anti-mouse IgG-HRP (anti-mouse IgG-peroxidase antibody produced in goat, sigma, catalog: A3673-1 ML) and anti-mouse IgG coating (anti-mouse IgG antibody produced in goat (Sigma, catalog: M2650-1 ML)) data were analyzed using FlowJo Software 10.7.1.
In vitro cytotoxicity assay
The cytotoxicity of CART cells was assessed using different effector cell ratios and at different time points. Target cells used in these assays were modified with lentiviral vectors to overexpress GFP-firefly luciferase (GFP-ffLuc), as described previously (Shah et al Antigen Presenting Cell-Mediated Expansion of Human Umbilical Cord Blood Yields Log-Scale Expansion of Natural Killer Cells with Anti-Myeloma Activity.PLoS One.2013;8(10)).
The percentage of remaining viable GFP+ tumor cells was analyzed by flow cytometry and calculated using the following formula: % = 100x for live cells (GFP+ cells with T cells at time x/GFP+ cells alone at time x).
In vitro proliferation assay
Proliferation of CART84 in response to CD84 antigen was measured using CFSE assay (Castella, m.et al (2019) mol. Ter. -Methods clin. Dev. 12:134-144). CART cells were stained with CELLTRACE CFSE (Invitrogen, thermosfisher, 15598431) and then co-cultured for 96 hours with or without stimulus. Proliferation was analyzed by flow cytometry and Proliferation Index (PI) (pi=sum of cell numbers in different generations/cell number) was calculated.
In vitro cytokine production
IFN-. Gamma., TNF-. Alpha., IL-6, IL-1. Beta. Cytokines were quantified by enzyme-linked immunosorbent assay (DuoSet ELISA, R & D system) according to the manufacturer's protocol.
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the disclosed antigen binding domains, antibodies, chimeric antigen receptors, uses, and methods will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been disclosed in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the disclosed modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the following claims.
Example 9: CD84CAR engineering
Several anti-CD 84CAR constructs were engineered based on antibodies GYCD 84.1.1-7, GYCD84.1-226, and GYCD 84.3.3-89. The complete CAR84 sequence, comprising the signal peptide, scFv specific for CD84, CD 8. Alpha. Hinge and transmembrane regions, co-stimulatory domain 4-1BB and signaling domain CD3- ζ, was cloned into a third generation lentiviral vector pCCL (Dull et al AThird-Generation Lentivirus Vector with a Conditional Packaging System. J. Virol. 1998:72:8463-8471) under the control of the EF 1. Alpha. Promoter (FIG. 6).
Two different scfvs were designed based on antibodies GYCD 84.1.1-7, differing in the order of the VH and VL sequences. All these CARs contain linkers with four (S4) motifs (Gly-Ser). The name assigned to each CAR version reflects the design of the scFv. The sequences of the different scFv domains synthesized and cloned in the pCCL vectors are listed in table 14.
Table 14. The sequence and structure of scFv domains used in each CD84CAR designed.
Example 10: expression of CD84 in hematological malignancies
The expression of CD84 on the surface of leukemia cells of patients diagnosed with AML or with T-ALL was analyzed by flow cytometry. Tables 15 and 16 summarize qualitative assessments of CD84 expression on AML and T-ALL cells, respectively. Fig. 5 shows two representative examples of flow cytometry analysis of primary leukemia cells of AML samples. FIG. 21 shows analysis of T-ALL samples (patient 01 and patient 02).
Table 15. Expression of CD84 on leukemia cells of 16 patients with AML was assessed by flow cytometry.
| Patient numbering | CD84 expression |
| P11 | Medium and medium |
| P12 | High height |
| P13 | Medium and medium |
| P14 | High height |
| P15 | Medium and medium |
| P16 | Medium and medium |
| P17 | High height |
| P18 | Medium and medium |
| P19 | Low and low |
| P20 | Medium and medium |
| P21 | Medium and medium |
| P22 | Low and low |
| P23 | High height |
| P24 | High height |
| P25 | High height |
| P26 | High height |
Table 16. Expression of CD84 on leukemia cells of two patients with T-ALL was assessed by flow cytometry.
| Patient numbering | CD84 expression |
| P01 | High height |
| P02 | High height |
Example 11: CD84CART Generation
Lentiviruses (LV) containing CART84 1.7.3, 1.226.5 and 3.89.5pCCL-EF 1. Alpha. -CD84 vectors were produced in HEK293-T cells and the number of transducing units per mL was determined by limiting dilution. Lentiviruses were used to transduce T cells isolated from whole blood, and these CAR-T cells were then expanded for 6 to 8 days.
Table 17 summarizes the number of T cells obtained after three different transduction, and the percentage of T cells expressing CD84CAR on their surface.
Table 17 number and percentage of CAR positive T cells obtained after 6-7 days transduction with lentivirus expressing CD84 CAR. Data from three independent transduction is shown.
Figure 22 shows further analysis of the amplification of CART84 1.7.3, 1.226.5 and 3.89.5 and of the amplification of 152.3, 153.5 and H3.5 6-7 days after transduction, expressed as a fold increase in T cell numbers relative to day 0.
Example 12: CD84CART in vitro cytotoxicity
Based on cytotoxicity results obtained for Ramos, K562, MOLM-13, NALM6 and MOLT-4 cells CART84 152.3, 153.5 and H3.5 were selected for additional characterization and compared to 1.7.3 and 1.226.5 and 3.89.5. The percent of viable GFP positive cells was determined by flow cytometry at 4:1, 2:1, 1:1 and 0.5:1 effector to target cell ratios, and their cytotoxicity against Ramos, MOLM-13, U937 and MOLT-4 cells after 24 or 48 hours co-culture was assessed. FIG. 3 shows CD84 expression on these cells.
All CART84 exerted a high cytotoxic effect against Ramos cells (CD 84 High height), which was statistically significant compared to UT (fig. 23). The cytotoxic effects of all CART cells increase over time; however, for H3.5 and 1.226.5, this effect is lower, especially at lower ratios (1:1 and 0.5:1).
CART84 152.3, 153.5, 1.7.3, 1.226.5 and 3.89.5 showed higher cytotoxic effects against MOLM-13 (CD 84 Medium and medium) and U937 (CD 84 Low and low) AML cells, statistically significant compared to UT (fig. 24 and 25, respectively). CART84H3.5 exert lower cytotoxic activity than other CART cells against leukemia cells from both cell lines. The cytotoxic effects of all CART increase over time. All CART cells, except H3.5, showed high cytotoxicity to U937 cells despite low CD84 expression.
Finally, cytotoxicity of CART84 152.3, 153.5, 1.7.5, 1.226.5 and 3.89.5 on MOLT-4 cells (CD 84 High height) was evaluated, the same pattern as U937 cells was observed, i.e. all CART cells exerted a high cytotoxic effect that increased over time, except for H3.5, and was statistically significant compared to UT (fig. 26).
Example 13: in vitro cytotoxicity of CD84CART against AML and T-ALL primary leukemia blasts
CART84 cells 152.3, 153.5, G7.5 and h.3.5 were evaluated for cytotoxicity against primary AML cells from patient samples obtained from Haematology Department of Hospital Cl i nic de Barcelona (HCB). CD84 expression was assessed on primary master cells, which were then stained with CFSE and co-cultured with effector cells (CART/UT) at effector to target cell ratios of 4:1, 2:1, 1:1 and 0.5:1 for 24h and 48h. After this period, cells were stained with LIVE/DeadTM Fixable Aqua to distinguish between LIVE/Dead cells. CART cells 152.3 and 153.5 exhibited the highest cytotoxic activity against AML blasts, statistically significant compared to the effect of UT. CART H3.5 and G7.5 exert lower cytotoxic effects. The cytotoxic effects induced by all CART increased over time (fig. 27).
CART84 cells 152.3, 153.5, 1.7.3, 1.226.5, and 3.89.5 were also evaluated for cytotoxicity against primary T-ALL patient samples obtained from Haematology Department of Hospital Cl i nic de Barcelona (HCB). The experiments were performed as described above for AML samples. CART 152.3, 153.5 and 1.7.3 were more cytotoxic than CART 1.226.5 and 3.89.5 (fig. 28).
Example 14: CD84CART in vitro cytokine production
To assess the ability of each CART cell to release cytokines, supernatants of effector-target cell co-cultures were collected. Ramos, MOLM-13 and MOLT-4 were used as target cells at a 2:1 effector cell to target cell ratio. After 24 hours of co-culture, the levels of IFN-gamma (FIG. 29), granzyme-B (FIG. 30) and TNF-alpha (FIG. 31) in the supernatant were determined by enzyme-linked immunosorbent assay (ELISA). The non-transduced T cells (UTs) were used as negative controls (co-cultured with target cells). Four independent experiments were performed. IFN-gamma and granzyme B are cytotoxic cytokines, and TNF-alpha is a pro-inflammatory cytokine. CART84H3.5, when co-cultured with Ramos, showed the highest cytokine production in all CART84 tested. CART84 1.7.3 has a cytokine profile similar to H3.5 after co-culture with MOLM-13 and MOLT-4 cells. All other CART cells tested produced similar levels of cytokines. These results indicate that both H3.5 and 1.7.3CART cells can have tonic (tonic) signaling, i.e., constitutive or chronic activation in the absence of ligand.
Example 15: CD84CART in vitro proliferation
To assess the ability of each CART84 cell to specifically proliferate upon antigen CD84 binding, a CFSE proliferation assay was performed. Briefly, CART cells were stained with CELLTRACETM CFSE on day 0. Four cases were tested: day 0 cells (UT or CART cells); cells cultured on day 4 with medium alone; cells stimulated with non-specific stimulus IL-2 (50 UI/mL) on day 4; cells stimulated with specific antigen (i.e., with MOLM-13 cells) on day 4 have an effector to target cell ratio of 0.5:1. CART84 152.3, 153.5 and 1.226.5 could be demonstrated to proliferate more when exposed to CD84 antigen than when incubated with medium alone or with IL-2 alone (figure 32). CART H3.5, 1.7.3 and 3.89.5, on the other hand, proliferate similarly upon stimulation with MOLM-13 or IL-2 stimulators, suggesting that these CART84 may exhibit tonic signaling.
Example 16: in vitro cytotoxicity of CD84CART against hematopoietic progenitor stem cells
CD84 is expressed on malignant cells from several hematological malignancies. Among these are AML, where CD84 expression is shared (to a lesser extent) between myeloid leukemia blast cells and Hematopoietic Progenitor Stem Cells (HPSCs). The same holds for CD33 and CD123, both of which are targets for the development of CART products for the treatment of AML. FIG. 33 shows the expression of CD84, CD33 and CD123 on HPSC from apheresis products.
To assess potential bone marrow toxicity of CART84, their cytotoxicity against HPSC was examined on CD34+ HPSC obtained from two different sources: cord blood units (CBU, from BST) and apheresis products from healthy donors that have been mobilized with G-CSF for allogeneic stem cell transplantation (also from BST). CD34+ cells from apheresis products represent an ideal model for studying bone marrow toxicity; however, it is more common to use CD34+ cells from CBU to study this type of mid-target/tumor toxicity.
Purified CD34+ cells from CBU were co-cultured with CART84 152.3, 153.5, 1.7.3, 1.229 and 3.89.5 at 4:1 and 2:1 effector to target cell ratios for 24 hours. CART84 152.3, 153.5, 1.7.3 and 1.226.5 exhibited low to moderate cytotoxic activity against CD34+ cells from CBU, with the cytotoxic activity of CART84 3.89.5 being highest. Fig. 34 summarizes the results from 5 independent experiments.
For cytotoxicity assays against CD34+ HPSC from apheresis products, 3 different donor buffy coats were used to generate CART84. Surprisingly, CART84 has relatively low cytotoxicity against these cells. Although CART cytotoxicity increased after 48 hours, UT cytotoxicity also increased and UT was not specific (fig. 35).
Example 17: CD84CART in vitro T cytotoxicity
Since CD84 is expressed on T cells, the potential toxicity of CART84 to T cells was investigated. T cells are isolated directly from the whole blood of the donor; half of them were activated and transduced to generate CART cells, and the other half were cryopreserved until CART cells were available (i.e. for 6-7 days). Cells were stained with CFSE and then co-cultured with CART 84. Fig. 36 shows the results of 5 independent experiments. CART84152.3, 153.5, 1.7.3 and 3.89.5 exert moderate cytotoxic effects on their own T cells. On the other hand, the cytotoxic activity induced by 1.226.5 was low and statistically insignificant compared to UT.
To further understand which specific T cell fractions are targeted by CART84, the effect on the following T cell subpopulations was analyzed: primary T cells, central memory T cells, effector memory T cells, and effector CD4/CD8T cells. Different T cell subsets were studied by flow cytometry: 1) Target T cells prior to assay and 2) surviving T cells after co-culture with different CART84, as shown in figure 37. It was observed that the central memory T cell fraction was most affected by CART84, especially by 152.3, 153.5 and 1.7.3, as expected, since CD84 is mainly expressed on memory T cells. In addition, all T cell subsets remained after co-culture with CART 84.
Example 18: in vivo efficacy of anti-CD 84CART in AML models
In three different experiments with NOD Scid Gamma (NSG) immunodeficient mice, several CART84 cells were tested for efficacy using MOLM-13AML cells modified to express GFP-firefly luciferase (GFPffLuc). CART84 with the best in vitro efficacy and safety profile was selected for in vivo testing: 152.3, 153.5, H3.5, 1.7.3 and 1.226.5.
In the first and second experiments, 1x 106 or 0.25x 106 MOLM-13GFP-ffLuc cells were injected intravenously (iv) into NSG mice on day 0, respectively (fig. 38 and 39). Six days later, 4x 106 to 5x 106 UTs, CART84 cells 152.3, 153.5, H3.5 or 1.7.3i.v. were injected into mice. Mice treated with CART 152.3, 153.5 and 1.7.3 had statistically significant increases in survival compared to mice treated with UT.
In the experiment shown in fig. 40, 1x 105 MOLM-13GFP-ffLuc cells were injected i.v. on day 0 and UT, CART 152.3, 153.5 and 1.226.5 were injected i.v. at doses of 5x 106 and 3x 106 cells, respectively, after two and eleven days. CART 152.3 and 153.5 control tumor progression by bioluminescence monitoring, which was statistically significant compared to mice treated with UT. Furthermore, mice treated with CART 152.3 and 153.5 had statistically significant increases in survival compared to mice treated with UT.
The bone marrow and spleen of the euthanized mice at the end of the experiment were collected and analyzed by flow cytometry to quantify the presence of tumor cells (MOLM-13-GFPffLuc), human CD3+ T cells or CART cells. In both experiments, CART84 treated mice had significantly lower tumor cell numbers in both bone marrow and spleen compared to UT treated mice (fig. 39C and 40C). Human T cells proliferated in the bone marrow and spleen of mice treated with UT, most likely due to xenograft versus host disease (xeno-GvHD) (fig. 39D and 40D). CART84 cells were found in both bone marrow and spleen, most prominent being CART84152.3 (fig. 39E and 40E).
In summary, 152.3 and 153.5 are able to continuously control AML disease progression and increase survival of treated animals.
Example 19: in vivo efficacy of CART84 in T-ALL model
In two different experiments in NOD Scidγ (NSG) mice, the efficacy of CART84 cells was tested using MOLT-4T-ALL cells modified to express GFP-ffLuc. The following CART84 was tested: 152.3, 153.5, 1.7.3 and 1.226.5.
In the experiment shown in FIG. 41, 0.75x 106 MOLT-4GFP-ffLuc cells were i.v. injected into NSG mice on day 0; five days later, 3x 106 CART or UT were injected i.v. CART84 152.3 controls tumor progression in a statistically significant manner compared to UT-treated mice by bioluminescence monitoring. Furthermore, mice treated with CART 152.3 and 153.5 had statistically significant increases in survival compared to mice treated with UT.
In the experiment shown in FIG. 42, 4X 105 MOLT-4GFP-ffLuc cells were injected i.v. on day 0, and 5X 106 CART or UT were injected i.v. 2 days later. Treatment with CART84 152.3 and 153.5 elicited statistically significant efficacy compared to UT in terms of both controlling tumor progression and increasing survival monitored by bioluminescence.
The bone marrow and spleen of euthanized mice at the end of the experiment were analyzed by flow cytometry to quantify the presence of tumor cells (MOLT-4), human CD3+ T cells and CART cells. In this case, there was no statistically significant difference in the number of tumor cells found in bone marrow and spleen from different groups of animals (fig. 42C). Human T cells proliferated in both bone marrow and spleen in UT-treated mice, likely due to xenograft versus host disease (xeno-GvHD) (fig. 42D). CART cells were found in both bone marrow and spleen, especially in the case of 152.3 cells (fig. 42E).
CART 84.3 and 153.5 were most effective in controlling T-ALL disease and prolonging survival in two T-ALL experiments.
Example 20: in vitro toxicity of CD84CART on human primary cells
To assess potential in-target/tumor-free toxicity, CD84 expression by human primary cells was investigated, along with the potential cytotoxic effects induced subsequently by CART 84. The following human cells were studied: human coronary endothelial cells (HCAEC), human small airway epithelial cells (HsaEpC), human cardiac muscle cells (HCM), human kidney epithelial cells (HREpC), and Human Uterine Fibroblasts (HUF). Using flow cytometry, it was possible to demonstrate that CD84 was not expressed on the surface of these cells (fig. 43).
In the XCELLigence assay CART84 152.3 and 153.5 were found not to be cytotoxic to HsaEpC, HCM, HREpC and HUF. However, despite its lack of CD84 expression, some cytotoxic effects on HCAEC were observed, indicating that this effect is not antigen-specific. To confirm this, the cytotoxic activity of CART84 was compared to that of CD 123-targeted CART, as the antigen was expressed on the surface of endothelial cells. The cytotoxicity induced by CD 123-directed CART cells was significantly higher than that induced by CART84 152.3 or 153.5 (fig. 44).
Example 21: methods for examples 9 to 20
CD123CAR engineering
An anti-CD 123CAR construct was designed based on IL3scfv-IgG4 (L235E) -CD28gg- ζ (2692) CAR from US 2014/0271582. The CAR sequence, including the signal peptide (GMCSFR α), 26292scFv (VH-linker-VL), the IgG4-Fc hinge and CD28 transmembrane regions, the co-stimulatory domain CD28 and the signaling domain CD3- ζ, was cloned into a third generation lentiviral vector pCCL (Dull et al AThird-Generation Lentivirus Vector with a Conditional Packaging System J.Virol.1998:72:8463-8471) under the control of the EF1 α promoter.
Flow cytometry
Positive CAR fractions of T cells were detected with biotin-SP (long spacer) AffiniPure goat anti-mouse IgG, F (ab ') 2 fragment goat anti-mouse IgG (Jackson ImmunoResearch) or with biotin SP conjugated AffiniPure F (ab') 2 fragment goat anti-human IgG (Jackson ImmunoResearch), washed and then with BV421/PE conjugated streptavidin (eBioscience) and incubated with antibodies required to study each set of proteins as described below.
The following mabs to human proteins were used to study CAR T cell phenotype: CD197-BV510 (CCR 7), CD62L-FITC, CD4-Pecy7, CD8-APCH7, CD45RA-APC (Becton Dickinson).
The following mAbs to human proteins were used to study different populations of cells present in AML/T-ALL samples ：CD11-APC、CD19-Fitc、HLA-DR-A450、CD45-PerCPCy5.5、CD34-PerCPCy5.5、CD3-APC、CD3-APCH7、CD33-Fitc、CD14-Fitc、CD14-APCH7、CD13-PE、CD13-BV421(Becton Dickinson)、CD84-APC、CD84-PE(BioLegend)、CD45-A750、CD117-PeCy7(BeckmanCoulter)、CD123-PECy7(eBiosciences).
The following mAbs against human proteins were used to study the different monocyte populations ：CD3-BV421、CD14-APCH7、CD19-FITC、CD33-APC、CD34-PerCPCy5.5、CD38-APC、CD84-PE(BioLegend)、CD123-PECy7(eBiosciences) and HLA-DR-BV450 (Becton Dickinson) present in peripheral blood
The following mAbs against proteins were used for in vivo experiments performed in the MOLM-13 model: anti-human CD3-APCH, anti-human CD33-APC, anti-human CD45-PerCP-Cy5.5, anti-mouse CD45-PECy7, streptavidin-BV 421 (Becton Dickinson). The following mAbs against proteins were used for in vivo experiments performed in the MOLT-4 model: anti-human CD3-APC, anti-human CD45-PerCP-Cy5.5, anti-mouse CD45Pe-Cy7, streptavidin-BV 421 (Becton Dickinson).
All samples were run by BD FACSCantoTMII,LSRFortessaTM, L (DB Biosciences) or AttuneTM NxT (Invitrogen) flow cytometer and analyzed using FlowJo v10.8.0software (BD Biosciences).
In vitro cytotoxicity assays against primary leukemia blasts from AML and T-ALL patients
Primary master cells were stained with CFSE (ThermoFisher) and co-cultured with effector cells (CART/UT) at effector to target cell ratios of 4:1, 2:1, 1:1 and 0.5:1 for 24 hours and 48 hours. After this period, the co-cultured cells were stained with LIVE/DeadTM Fixable Aqua (ThermoFisher) to distinguish between LIVE/Dead cells. The percentage of remaining viable parent cells was determined by flow cytometry and calculated using the following formula: % = 100x of live cells (number of negative live/dead positive CFSE cells with CART cells at time x/number of negative live/dead positive CFSE cells alone at time x).
Isolation of CD34+ cells
Hematopoietic progenitor stem cells (CD 34+) were obtained from two sources: cord blood units (CBU, from blood bank Banc de Sang i Teixits de Barcelona, BST) and apheresis products from healthy donors that have been mobilized with G-CSF for allogeneic stem cell transplantation (also obtained from BST).
CD34+ cells were first isolated from CBU by Ficoll-Paque density gradient centrifugation, and then cd34+ hematopoietic progenitor stem cells were purified by magnetic separation using a human CD34-MicroBead kit (Miltenyi Biotec, positive selection). To isolate CD34+ cells from the apheresis product, the cells were directly purified using CD 34-beads.
In vitro cytotoxicity assays against CD34+ cells
To assess cytotoxicity of CD84CART on CD34+ cells, CD34+ cells were co-cultured with effector cells (CART/UT) at 4:1, 2:1 effector to target cell ratios for 24 hours and 48 hours. After this, the co-cultured cells were stained with APC-conjugated CD3 and PE-conjugated CD34 antibodies to distinguish effector cells from target cells, and LIVE/dead cells were distinguished with LIVE/DeadFixable Aqua.
In vitro cytotoxicity assays against purified T cells
T cells were isolated from the donor buffy coat during Ficoll-Paque density gradient centrifugation using immune cell isolation reagents from rosetteepTM. Half of the T cells obtained in the process are activated and transduced to generate CART cells, and the other half are cryopreserved for use as target cells until CART cells are available (i.e., for 8 days). For cytotoxicity assays, T cells were thawed and stained with CFSE, and then co-cultured with CART cells at different effector to target cell ratios. After 24 hours, both effector and target cells were fixed and stained with the LIV/Dead Fixable Aqua cell staining kit.
The percentage of T cells remaining was determined by flow cytometry and calculated using the following formula: % = 100x of live cells (number of negative live/dead positive CFSE cells with CART cells at time x/number of negative live/dead positive CFSE cells alone at time x).
To study the remaining subpopulations of live T cells, these cells were also stained with human mabs from the phenotype group as described in the flow cytometry section.
In vivo efficacy assay
Non-obese diabetic-Cg-PRKDCSCID IL2rgtm1Wjl/SzJ (NSG) mice (CHARLES RIVER) of eight weeks to 12 weeks of age were fed and housed under pathogen-free conditions in animal facilities at the university of barcelona medical and health sciences institute (Faculty of MEDICINE AND HEALTH SCIENCES of University of Barcelona). From 0.1X106 to 1X 106 MOLM-13 or GFPffLuc-MOLT-4 cells expressing GFPffLuc were suspended in saline and injected Intravenously (IV) into each NSG mouse on day 0.3 x 106 to 5 x 106 UTs or CD84CART are infused intravenously (i.v.) 2 to 9 days after tumor cell infusion. Tumor progression was monitored by bioluminescence using a Xenogen IVIS 50Imaging System (Perkinelmer). To measure bioluminescence, 100uL XenoLight D-Luciferin (PERKIN ELMER ref.122799) was administered intraperitoneally into each mouse and the tumor burden was monitored once a week. The total luminous flux was visualized and calculated using LIVING IMAGE software (PerkinElmer). Mice were euthanized when signs of humane endpoint criteria were presented. Bone marrow and spleen were extracted and analyzed by flow cytometry to quantify the presence of tumor cells, T cells and CART cells.
All procedures were performed in compliance with the institutional animal care committee of the university of bastarna medical science and the health sciences.
Primary human cells
The following primary human cells were obtained from PromoCell: human coronary endothelial cells (HCAEC), human small airway epithelial cells (HSAEpC), human Uterine Fibroblasts (HUF), human myocardial cells (HCM), and human kidney epithelial cells (HREpC). Cells were cultured at 37 ℃ and 5% co2 using specific media and supplements (also from PromoCells) as shown in their data table.
In vitro cytotoxicity assay using adherent cells
To assess CART cytotoxicity to adherent cells, an xcelligent instrument was used that allowed live cell proliferation to be measured in real time by impedance. Target cells were seeded into RTCAE-Plates (16 wells) of xcelligent and cell index was monitored over a 24h period using xcelligent RTCA multi-plate monitoring system. At this point, 100mL of growth medium or T cells (untransduced T cells or CART cells at 4:1 effector cells to target cell ratio) were added to each well. The data (cell index) obtained from each well was normalized to 1 and cell growth monitored during 72 h. Data were analyzed using xCELLigence RTCASoftware Lite 2.0.0.1301.
Example 22: in vivo efficacy of CART84 in B-cell lymphoma model
In vivo experiments in NOD Scid Gamma (NSG) mice, several CART84 cells were tested for efficacy using Ramos (burkitt lymphoma) cells modified to express GFP-ffLuc. The following CART84 was tested: 152.3, 153.5 and H3.5.
In the experiment shown in fig. 45, 4x 105 Ramos GFP-ffLuc cells were i.v. injected into NSG mice on day 0. Two days later, 5x 106 CART or UT cells were injected i.v. Mice treated with CART 152.3, 153.5 and H3.5 showed a tendency to grow slower (fig. 45A). Bone marrow from euthanized mice at the end of the experiment was collected and analyzed by flow cytometry to quantify the presence of tumor cells. CART84 treated mice had statistically significantly lower numbers of Ramos GFP-ffLuc cells in bone marrow when compared to UT treated mice (fig. 45B).