VISTA ANTIGEN-BINDING MOLECULES
20210380697 · 2021-12-09
Assignee
Inventors
- Jerome Douglas Boyd-Kirkup (Singapore, SG)
- Piers Ingram (Singapore, SG)
- Dipti Thakkar (Singapore, SG)
- Zhihao Wu (Singapore, SG)
- Konrad Paszkiewicz (Singapore, SG)
- Vicente Sancenon (Singapore, SG)
- Siyu Guan (Singapore, SG)
Cpc classification
C07K2317/72
CHEMISTRY; METALLURGY
C07K2317/33
CHEMISTRY; METALLURGY
C07K2317/94
CHEMISTRY; METALLURGY
A61K2039/507
HUMAN NECESSITIES
C07K2317/24
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
C07K16/28
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
Abstract
VISTA antigen-binding molecules are disclosed. Also disclosed are nucleic acids and expression vectors encoding, compositions comprising, and methods using, the VISTA antigen-binding molecules.
Claims
1. An antigen-binding molecule, optionally isolated, which is capable of binding to VISTA and inhibiting VISTA-mediated signalling, independently of Fc-mediated function.
2. The antigen-binding molecule according to claim 1, wherein the antigen-binding molecule comprises: (i) a heavy chain variable (VH) region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:305 HC-CDR2 having the amino acid sequence of SEQ ID NO:306 HC-CDR3 having the amino acid sequence of SEQ ID NO:307; and (ii) a light chain variable (VL) region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO:41 LC-CDR2 having the amino acid sequence of SEQ ID NO:308 LC-CDR3 having the amino acid sequence of SEQ ID NO:43.
3. The antigen-binding molecule according to claim 1 or claim 2, wherein the antigen-binding molecule comprises: (i) a heavy chain variable (VH) region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:290 HC-CDR2 having the amino acid sequence of SEQ ID NO:291 HC-CDR3 having the amino acid sequence of SEQ ID NO:278; and (ii) a light chain variable (VL) region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO:41 LC-CDR2 having the amino acid sequence of SEQ ID NO:309 LC-CDR3 having the amino acid sequence of SEQ ID NO:43.
4. The antigen-binding molecule according to any one of claims 1 to 3, wherein the antigen-binding molecule comprises: (i) a heavy chain variable (VH) region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:290 HC-CDR2 having the amino acid sequence of SEQ ID NO:291 HC-CDR3 having the amino acid sequence of SEQ ID NO:278; and (ii) a light chain variable (VL) region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO:41 LC-CDR2 having the amino acid sequence of SEQ ID NO:295 LC-CDR3 having the amino acid sequence of SEQ ID NO:43.
5. The antigen-binding molecule according to any one of claims 1 to 3, wherein the antigen-binding molecule comprises: (i) a heavy chain variable (VH) region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO:290 HC-CDR2 having the amino acid sequence of SEQ ID NO:291 HC-CDR3 having the amino acid sequence of SEQ ID NO:278; and (ii) a light chain variable (VL) region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO:41 LC-CDR2 having the amino acid sequence of SEQ ID NO:300 LC-CDR3 having the amino acid sequence of SEQ ID NO:43.
6. The antigen-binding molecule according to any one of claims 1 to 5, wherein the antigen-binding molecule comprises: a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:289; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:310.
7. The antigen-binding molecule according to any one of claims 1 to 6, wherein the antigen-binding molecule comprises: a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:289; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:294.
8. The antigen-binding molecule according to any one of claims 1 to 6, wherein the antigen-binding molecule comprises: a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:289; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:297.
9. The antigen-binding molecule according to any one of claims 1 to 6, wherein the antigen-binding molecule comprises: a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:289; and a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:299.
10. An antigen-binding molecule, optionally isolated, comprising (i) an antigen-binding molecule according to any one of claims 1 to 9, and (ii) an antigen-binding molecule capable of binding to an antigen other than VISTA.
11. A chimeric antigen receptor (CAR) comprising an antigen-binding molecule according to any one of claims 1 to 10.
12. A nucleic acid, or a plurality of nucleic acids, optionally isolated, encoding an antigen-binding molecule according to any one of claims 1 to 10 or a CAR according to claim 11.
13. An expression vector, or a plurality of expression vectors, comprising a nucleic acid or a plurality of nucleic acids according to claim 12.
14. A cell comprising an antigen-binding molecule according to any one of claims 1 to 10, a CAR according to claim 11, a nucleic acid or a plurality of nucleic acids according to claim 12, or an expression vector or a plurality of expression vectors according to claim 13.
15. A method comprising culturing a cell comprising a nucleic acid or a plurality of nucleic acids according to claim 12, or an expression vector or a plurality of expression vectors according to claim 13, under conditions suitable for expression of the antigen-binding molecule or CAR from the nucleic acid(s) or expression vector(s).
16. A composition comprising an antigen-binding molecule according to any one of claims 1 to 10, a CAR according to claim 11, a nucleic acid or a plurality of nucleic acids according to claim 12, an expression vector or a plurality of expression vectors according to claim 13, or a cell according to claim 14.
17. The composition according to claim 16, additionally comprising an agent capable of inhibiting signalling mediated by an immune checkpoint molecule other than VISTA, optionally wherein the immune checkpoint molecule other than VISTA is selected from PD-1, CTLA-4, LAG-3, TIM-3, TIGIT and BTLA.
18. An antigen-binding molecule according to any one of claims 1 to 10, a CAR according to claim 11, a nucleic acid or a plurality of nucleic acids according to claim 12, an expression vector or a plurality of expression vectors according to claim 13, a cell according to claim 14, or a composition according to claim 16 or claim 17 for use in a method of medical treatment or prophylaxis.
19. An antigen-binding molecule according to any one of claims 1 to 10, a CAR according to claim 11, a nucleic acid or a plurality of nucleic acids according to claim 12, an expression vector or a plurality of expression vectors according to claim 13, a cell according to claim 14, or a composition according to claim 16 or claim 17, for use in a method of treatment or prevention of a cancer or an infectious disease.
20. Use of an antigen-binding molecule according to any one of claims 1 to 10, a CAR according to claim 11, a nucleic acid or a plurality of nucleic acids according to claim 12, an expression vector or a plurality of expression vectors according to claim 13, a cell according to claim 14, or a composition according to claim 16 or claim 17, in the manufacture of a medicament for use in a method of treatment or prevention of a cancer or an infectious disease.
21. A method of treating or preventing a cancer or an infectious disease, comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen-binding molecule according to any one of claims 1 to 10, a CAR according to claim 11, a nucleic acid or a plurality of nucleic acids according to claim 12, an expression vector or a plurality of expression vectors according to claim 13, a cell according to claim 14, or a composition according to claim 16 or claim 17.
22. The antigen-binding molecule, CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell or composition for use according to claim 19, the use according to claim 20 or the method according to claim 21, wherein the cancer is selected from: a cancer comprising cells expressing VISTA, a cancer comprising infiltration of cells expressing VISTA, a cancer comprising cancer cells expressing VISTA, a hematological cancer, leukemia, acute myeloid leukemia, lymphoma, B cell lymphoma, T cell lymphoma, multiple myeloma, mesothelioma, a solid tumor, lung cancer, non-small cell lung carcinoma, gastric cancer, gastric carcinoma, colorectal cancer, colorectal carcinoma, colorectal adenocarcinoma, uterine cancer, uterine corpus endometrial carcinoma, breast cancer, triple negative breast invasive carcinoma, liver cancer, hepatocellular carcinoma, pancreatic cancer, pancreatic ductal adenocarcinoma, thyroid cancer, thymoma, skin cancer, melanoma, cutaneous melanoma, kidney cancer, renal cell carcinoma, renal papillary cell carcinoma, head and neck cancer, squamous cell carcinoma of the head and neck (SCCHN), ovarian cancer, ovarian carcinoma, ovarian serous cystadenocarcinoma, prostate cancer and/or prostate adenocarcinoma.
23. An antigen-binding molecule according to any one of claims 1 to 10, a CAR according to claim 11, a nucleic acid or a plurality of nucleic acids according to claim 12, an expression vector or a plurality of expression vectors according to claim 13, a cell according to claim 14, or a composition according to claim 16 or claim 17, for use in a method of treatment or prevention of a disease in which myeloid-derived suppressor cells (MDSCs) are pathologically implicated.
24. Use of an antigen-binding molecule according to any one of claims 1 to 10, a CAR according to claim 11, a nucleic acid or a plurality of nucleic acids according to claim 12, an expression vector or a plurality of expression vectors according to claim 13, a cell according to claim 14, or a composition according to claim 16 or claim 17, in the manufacture of a medicament for use in a method of treatment or prevention of a disease in which myeloid-derived suppressor cells (MDSCs) are pathologically implicated.
25. A method of treating or preventing a disease in which myeloid-derived suppressor cells (MDSCs) are pathologically implicated, comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen-binding molecule according to any one of claims 1 to 10, a CAR according to claim 11, a nucleic acid or a plurality of nucleic acids according to claim 12, an expression vector or a plurality of expression vectors according to claim 13, a cell according to claim 14, or a composition according to claim 16 or claim 17.
26. The antigen-binding molecule, CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell or composition for use, the use, or the method according to any one of claims 19 to 25, wherein the method additionally comprises administration of an agent capable of inhibiting signalling mediated by an immune checkpoint inhibitor other than VISTA, optionally wherein the immune checkpoint inhibitor other than VISTA is selected from PD-1, CTLA-4, LAG-3, TIM-3, TIGIT or BTLA.
27. A method of inhibiting VISTA-mediated signalling, comprising contacting VISTA-expressing cells with an antigen-binding molecule according to any one of claims 1 to 10.
28. A method for inhibiting the activity of myeloid-derived suppressor cells (MDSCs), the method comprising contacting MDSCs with an antigen-binding molecule according to any one of claims 1 to 10.
29. A method for increasing the number or activity of effector immune cells, the method comprising inhibiting the activity of VISTA-expressing cells with an antigen-binding molecule according to any one of claims 1 to 10.
30. An in vitro complex, optionally isolated, comprising an antigen-binding molecule according to any one of claims 1 to 10 bound to VISTA.
31. A method comprising contacting a sample containing, or suspected to contain, VISTA with an antigen-binding molecule according to any one of claims 1 to 10, and detecting the formation of a complex of the antigen-binding molecule with VISTA.
32. A method of selecting or stratifying a subject for treatment with a VISTA-targeted agent, the method comprising contacting, in vitro, a sample from the subject with an antigen-binding molecule according to any one of claims 1 to 10 and detecting the formation of a complex of the antigen-binding molecule with VISTA.
33. Use of an antigen-binding molecule according to any one of claims 1 to 10 as an in vitro or in vivo diagnostic or prognostic agent.
34. Use of an antigen-binding molecule according to any one of claims 1 to 10 in a method for detecting, localizing or imaging a cancer, optionally wherein the cancer is selected from: a cancer comprising cells expressing VISTA, a cancer comprising infiltration of cells expressing VISTA, a cancer comprising cancer cells expressing VISTA, a hematological cancer, leukemia, acute myeloid leukemia, lymphoma, B cell lymphoma, T cell lymphoma, multiple myeloma, mesothelioma, a solid tumor, lung cancer, non-small cell lung carcinoma, gastric cancer, gastric carcinoma, colorectal cancer, colorectal carcinoma, colorectal adenocarcinoma, uterine cancer, uterine corpus endometrial carcinoma, breast cancer, triple negative breast invasive carcinoma, liver cancer, hepatocellular carcinoma, pancreatic cancer, pancreatic ductal adenocarcinoma, thyroid cancer, thymoma, skin cancer, melanoma, cutaneous melanoma, kidney cancer, renal cell carcinoma, renal papillary cell carcinoma, head and neck cancer, squamous cell carcinoma of the head and neck (SCCHN), ovarian cancer, ovarian carcinoma, ovarian serous cystadenocarcinoma, prostate cancer and/or prostate adenocarcinoma.
Description
BRIEF DESCRIPTION OF THE FIGURES
[1435] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures.
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EXAMPLES
[1502] In the following Examples, the inventors describe the generation of novel anti-VISTA antibody clones targeted to specific regions of interest in the VISTA molecule, and the biophysical and functional characterisation and therapeutic evaluation of these antigen-binding molecules.
Example 1: VISTA Target Design and Anti-VISTA Antibody Hybridoma Production
[1503] The inventors selected regions in the extracellular region of human VISTA (SEQ ID NO:3) for raising VISTA-binding monoclonal antibodies.
[1504] The FG loop region was targeted because this region of VISTA has been proposed to be important for VISTA's inhibitory function (Vigdorovich et al., Structure. 2013; 21(5):707-717). The front-facing β-sheet region of VISTA was also targeted.
1.1 Hybridoma production
[1505] Approximately 6 week old female BALB/c mice were obtained from InVivos (Singapore). Animals were housed under specific pathogen-free conditions and were treated in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines.
[1506] For hybridoma production, mice were immunized with proprietary mixtures of antigenic peptide, recombinant target protein or cells expressing the target protein.
[1507] Prior to harvesting the spleen for fusion, mice were boosted with antigen mixture for three consecutive days. 24 h after the final boost total splenocytes were isolated and fused with the myeloma cell line P3X63.Ag8.653 (ATCC, USA), with PEG using ClonaCell-HY Hybridoma Cloning Kit, in accordance with the manufacturer's instructions (Stemcell Technologies, Canada).
[1508] Fused cells were cultured in ClonaCell-HY Medium C (Stemcell Technologies, Canada) overnight at 37° C. in a 5% CO.sub.2 incubator. The next day, fused cells were centrifuged and resuspended in 10 ml of ClonaCell-HY Medium C and then gently mixed with 90 ml of semisolid methylcellulose-based ClonaCell-HY Medium D (StemCell Technologies, Canada) containing HAT components, which combines the hybridoma selection and cloning into one step.
[1509] The fused cells were then plated into 96 well plates and allowed to grow at 37° C. in a 5% CO.sub.2 incubator. After 7-10 days, single hybridoma clones were isolated and antibody producing hybridomas were selected by screening the supernatants by Enzyme-linked immunosorbent assay (ELISA) and Fluorescence-activated cell sorting (FACs).
1.2 Antibody variable region amplification and sequencing
[1510] Total RNA was extracted from hybridoma cells using TRIzol reagent (Life Technologies, Inc., USA) using manufacturer's protocol. Double-stranded cDNA was synthesized using SMARTer RACE 5′/3′ Kit (Clontech™, USA) in accordance with the manufacturer's instructions. Briefly, 1 μg total RNA was used to generate full-length cDNA using 5′-RACE CDS primer (provided in the kit), and the 5′ adaptor (SMARTer II A primer) was then incorporated into each cDNA according to manufacturers instructions. cDNA synthesis reactions contained: 5× First-Strand Buffer, DTT (20 mM), dNTP Mix (10 mM), RNase Inhibitor (40 U/μl) and SMARTScribe Reverse Transcriptase (100 U/μl).
[1511] The race-ready cDNAs were amplified using SeqAmp DNA Polymerase (Clontech™, USA). Amplification reactions contained SeqAmp DNA Polymerase, 2× Seq AMP buffer, 5′ universal primer provided in the 5′ SMARTer Race kit, that is complement to the adaptor sequence, and 3′ primers that anneal to respective heavy chain or light chain constant region primer. The 5′ constant region were designed based on previously reported primer mix either by Krebber et al. J. Immunol. Methods 1997; 201: 35-55, Wang et al. Journal of Immunological Methods 2000, 233; 167-177 or Tiller et al. Journal of Immunological Methods 2009; 350:183-193. The following thermal protocol was used: pre-denature cycle at 94° C. for 1 min; 35 cycles of 94° C., 30 s, 55° C., 30 s and 72° C., 45 s; final extension at 72° C. for 3 min.
[1512] The resulting VH and VL PCR products, approximately 550 bp, were cloned into pJET1.2/blunt vector using CloneJET PCR Cloning Kit (Thermo Scientific, USA) and used to transform highly competent E. coli DH5a. From the resulting transformants, plasmid DNA was prepared using Miniprep Kit (Qiagene, Germany) and sequenced. DNA sequencing was carried out by AITbiotech. These sequencing data were analyzed using the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue):D413-22) to characterize the individual CDRs and framework sequences. The signal peptide at 5′ end of the VH and VL was identified by SignalP (v 4.1; Nielsen, in Kihara, D (ed): Protein Function Prediction (Methods in Molecular Biology vol. 1611) 59-73, Springer 2017).
[1513] Monoclonal anti-VISTA antibody clones were then selected for further development and characterisation. Humanised versions of antibody clone 4M2-C12 (also referred to herein as “V4”) were also prepared according to standard methods by cloning the CDRs of antibodies into VH and VL comprising human antibody framework regions.
TABLE-US-00003 Peptide immunogen used Antibody clone VH/VLsequence to raise the antibody 4M2-C12 (also VH = SEQ ID NO: 32 SEQ ID NO: 26 referred to herein as VL = SEQ ID NO: 40 “V4”) V4H1 VH = SEQ ID NO: 52 VL = SEQ ID NO: 57 V4H2 VH = SEQ ID NO: 62 VL = SEQ ID NO: 66 4M2-B4 VH = SEQ ID NO: 48 VL = SEQ ID NO: 50 4M2-C9 VH = SEQ ID NO: 87 VL = SEQ ID NO: 95 4M2-D9 VH = SEQ ID NO: 106 VL = SEQ ID NO: 113 4M2-D5 VH = SEQ ID NO: 143 VL = SEQ ID NO: 150 4M2-A8 VH = SEQ ID NO: 157 VL = SEQ ID NO: 164 2M1-B12 VH = SEQ ID NO: 71 SEQ ID NO: 27 VL = SEQ ID NO: 79 2M1-D2 VH = SEQ ID NO: 102 VL = SEQ ID NO: 104 1M2-D2 VH = SEQ ID NO: 119 SEQ ID NO: 28 VL = SEQ ID NO: 126 13D5p VH = SEQ ID NO: 183 VL = SEQ ID NO: 188 13D5-1 VH = SEQ ID NO: 194 VL = SEQ ID NO: 196 13D5-13 VH = SEQ ID NO: 199 VL = SEQ ID NO: 202 5M1-A11 VH = SEQ ID NO: 133 SEQ ID NO: 29 VL = SEQ ID NO: 136 9M2-C12 VH = SEQ ID NO: 168 SEQ ID NO: 30 VL = SEQ ID NO: 176
Example 2: Antibody Production and Purification
[1514] 2.1 Cloning VH and VL into Expression Vectors:
[1515] DNA sequences encoding the heavy and light chain variable regions of the anti-VISTA antibody clones were subcloned into the pmAbDZ_IgG1_CH and pmAbDZ_IgG1_CL (InvivoGen, USA) eukaryotic expression vectors for construction of human-mouse chimeric antibodies.
[1516] Alternatively, DNA sequence encoding the heavy and light chain variable regions of the anti-VISTA antibody clones were subcloned into the pFUSE-CHIg-hG1 and pFUSE2ss-CLIg-hk (InvivoGen, USA) eukaryotic expression vectors for construction of human-mouse chimeric antibodies. Human IgG1 constant region encoded by pFUSE-CHIg-hG1 comprises the substitutions D356E, L358M (positions numbered according to EU numbering) in the CH3 region relative to Human IgG1 constant region (IGHG1; UniProt:P01857-1, v1; SEQ ID NO:210). pFUSE2ss-CLIg-hk encodes human IgG1 light chain kappa constant region (IGCK; UniProt: P01834-1, v2).
[1517] Variable regions along with the signal peptides were amplified from the cloning vector using SeqAmp enzyme (Clontech™, USA) following the manufacturer's protocol. Forward and reverse primers having 15-20 bp overlap with the appropriate regions within VH or VL plus 6 bp at 5′ end as restriction sites were used. The DNA insert and the pFuse vector were digested with restriction enzyme recommended by the manufacturer to ensure no frameshift was introduced (e.g., EcoRI and NheI for VH, AgeI and BsiWI for VL,) and ligated into its respective plasmid using T4 ligase enzyme (Thermo Scientific, USA). The molar ratio of 3:1 of DNA insert to vector was used for ligation.
2.2 Expression of antibodies in mammalian cells
[1518] Antibodies were expressed using either 1) Expi293 Transient Expression System Kit (Life Technologies, USA), or 2) HEK293-6E Transient Expression System (CNRC-NRC, Canada) following the manufacturer's instructions.
1) Expi293 Transient Expression System:
Cell Line Maintenance:
[1519] HEK293F cells (Expi293F) were obtained from Life Technologies, Inc (USA). Cells were cultured in serum-free, protein-free, chemically defined medium (Expi293 Expression Medium, Thermo Fisher, USA), supplemented with 50 Um/ml penicillin and 50 μg/ml streptomycine (Gibco, USA) at 37° C., in 8% CO.sub.2 and 80% humidified incubators with shaking platform.
Transfection:
[1520] Expi293F cells were transfected with expression plasmids using ExpiFectamine 293 Reagent kit (Gibco, USA) according to its manufacturer's protocol. Briefly, cells at maintenance were subjected to a media exchange to remove antibiotics by spinning down the culture, cell pellets were re-suspended in fresh media without antibiotics at 1 day before transfection. On the day of transfection, 2.5×10.sup.6/ml of viable cells were seeded in shaker flasks for each transfection. DNA-ExpiFectamine complexes were formed in serum-reduced medium, Opti-MEM (Gibco, USA), for 25 min at room temperature before being added to the cells. Enhancers were added to the transfected cells at 16-18 h post transfection. An equal amount of media was topped up to the transfectants at day 4 post-transfection to prevent cell aggregation. Transfectants were harvested at day 7 by centrifugation at 4000×g for 15 min, and filtered through 0.22 μm sterile filter units.
2) HEK293-6E Transient Expression System
Cell Line Maintenance:
[1521] HEK293-6E cells were obtained from National Research Council Canada. Cells were cultured in serum-free, protein-free, chemically defined Freestyle F17 Medium (Invitrogen, USA), supplemented with 0.1% Kolliphor-P188 and 4 mM L-Glutamine (Gibco, USA) and 25 μg/ml G-418 at 37° C., in 5% CO.sub.2 and 80% humidified incubators with shaking platform.
Transfection:
[1522] HEK293-6E cells were transfected with expression plasmids using PElpro™ (Polyplus, USA) according to its manufacturer's protocol. Briefly, cells at maintenance were subjected to a media exchange to remove antibiotics by centrifugation, cell pellets were re-suspended with fresh media without antibiotics at 1 day before transfection. On the day of transfection, 1.5-2×10.sup.6 cells/ml of viable cells were seeded in shaker flasks for each transfection. DNA and PEIpro™ were mixed to a ratio of 1:1 and the complexes were allowed to form in F17 medium for 5 min at RT before adding to the cells. 0.5% (w/v) of Tryptone N1 was fed to transfectants at 24-48 h post transfection. Transfectants were harvested at day 6-7 by centrifugation at 4000×g for 15 min and the supernatant was filtered through 0.22 μm sterile filter units.
[1523] Cells were transfected with vectors encoding the following combinations of polypeptides:
TABLE-US-00004 Antigen- biding molecule Polypeptides Antibody [1] 4M2-C12 VH-CH1-CH2-CH3 anti-VISTA clone (SEQ ID NO: 212) + 4M2-C12 IgG1 4M2-C12 VL-Cκ (SEQ ID NO: 213) [2] 4M2-B4 VH-CH1-CH2-CH3 anti-VISTA clone (SEQ ID NO: 214) + 4M2-B4 IgG1 4M2-B4 VL-Cκ (SEQ ID NO: 215) [3] V4H1 VH-CH1-CH2-CH3 anti-VISTA clone (SEQ ID NO: 216) + V4H1 IgG1 V4H1 VL-Cκ (SEQ ID NO: 217) [4] V4H2 VH-CH1-CH2-CH3 anti-VISTA clone (SEQ ID NO: 218) + V4H2 IgG1 V4H2 VL-Cκ (SEQ ID NO: 219) [5] 2M1-B12 VH-CH1-CH2-CH3 anti-VISTA clone (SEQ ID NO: 220) + 2M1-B12 IgG1 2M1-B12 VL-Cκ (SEQ ID NO: 221) [6] 4M2-C9 VH-CH1-CH2-CH3 anti-VISTA clone (SEQ ID NO: 222) + 4M2-C9 IgG1 4M2-C9 VL-Cκ (SEQ ID NO: 223) [7] 2M1-D2 VH-CH1-CH2-CH3 anti-VISTA clone (SEQ ID NO: 224) + 2M1-D2 IgG1 2M1-D2 VL-Cκ (SEQ ID NO: 225) [8] 4M2-D9 VH-CH1-CH2-CH3 anti-VISTA clone (SEQ ID NO: 226) + 4M2-D9 IgG1 4M2-D9 VL-Cκ (SEQ ID NO: 227) [9] 1M2-D2 VH-CH1-CH2-CH3 anti-VISTA clone (SEQ ID NO: 228) + 1M2-D2 IgG1 1M2-D2 VL-Cκ (SEQ ID NO: 229) [10] 5M1-A11 VH-CH1-CH2-CH3 anti-VISTA clone (SEQ ID NO: 230) + 5M1-A11 IgG1 5M1-A11 VL-Cκ (SEQ ID NO: 231) [11] 4M2-D5 VH-CH1-CH2-CH3 anti-VISTA clone (SEQ ID NO: 232) + 4M2-D5 IgG1 4M2-D5 VL-Cκ (SEQ ID NO: 233) [12] 4M2-A8 VH-CH1-CH2-CH3 anti-VISTA clone (SEQ ID NO: 234) + 4M2-A8 IgG1 4M2-A8 VL-Cκ (SEQ ID NO: 235) [13] 9M2-C12 VH-CH1-CH2-CH3 anti-VISTA clone (SEQ ID NO: 236) + 9M2-C12 IgG1 9M2-C12 VL-Cκ (SEQ ID NO: 237) [14] 13D5p VH-CH1-CH2-CH3 anti-VISTA clone (SEQ ID NO: 238) + 13D5p IgG1 13D5p VL-Cκ (SEQ ID NO: 239) [15] 13D5-1 VH-CH1-CH2-CH3 anti-VISTA clone (SEQ ID NO: 240) + 13D5-1 IgG1 13D5-1 VL-Cκ (SEQ ID NO: 241) [16] 13D5-13 VH-CH1-CH2-CH3 anti-VISTA clone (SEQ ID NO: 242) + 13D5-13 IgG1 13D5-13 VL-Cκ (SEQ ID NO: 243)
2.3 Antibody Purification
[1524] Affinity purification, buffer exchange and storage:
[1525] Antibodies secreted by the transfected cells into the culture supernatant were purified using liquid chromatography system AKTA Start (GE Healthcare, UK). Specifically, supernatants were loaded onto HiTrap Protein G column (GE Healthcare, UK) at a binding rate of 5 ml/min, followed by washing the column with 10 column volumes of washing buffer (20 mM sodium phosphate, pH 7.0). Bound mAbs were eluted with elution buffer (0.1 M glycine, pH 2.7) and the eluents were fractionated to collection tubes which contain appropriate amount of neutralization buffer (1 M Tris, pH 9). Neutralised elution buffer containing purified mAb were exchanged into PBS using 30K MWCO protein concentrators (Thermo Fisher, USA) or 3.5K MWCO dialysis cassettes (Thermo Fisher, USA). Monoclonal antibodies were sterilized by passing through 0.22 μm filter, aliquoted and snap-frozen in −80° C. for storage.
2.4 Antibody-purity analysis
Size exclusion chromatography (SEC):
[1526] Antibody purity was analysed by size exclusion chromatography (SEC) using Superdex 200 10/30 GL columns (GE Healthcare, UK) in PBS running buffer, on a AKTA Explorer liquid chromatography system (GE Healthcare, UK). 150 μg of antibody in 500 μl PBS pH 7.2 was injected to the column at a flow rate of 0.75 ml/min at room temperature. Proteins were eluted according to their molecular weights.
[1527] The result for anti-VISTA antibody clone V4 ([1] of Example 2.2) is shown in
Sodium-Dodecyl Sulfate Polyacrylamide gel electrophoresis (SDS-PAGE):
[1528] Antibody purity was also analysed by SDS-PAGE under reducing and non-reducing conditions according to standard methods. Briefly, 4%-20% TGX protein gels (Bio-Rad, USA) were used to resolve proteins using a Mini-Protean Electrophoresis System (Bio-Rad, USA). For non-reducing condition, protein samples were denatured by mixing with 2× Laemmli sample buffer (Bio-Rad, USA) and boiled at 95° C. for 5-10 min before loading to the gel. For reducing conditions, 2× sample buffer containing 5% of β-mercaptoethanol (βME), or 40 mM DTT (dithiothreitol) was used. Electrophoresis was carried out at a constant voltage of 150V for 1 h in SDS running buffer (25 mM Tris, 192 mM glycine, 1% SDS, pH 8.3).
Western Blot:
[1529] Protein samples (30 μg) were fractionated by SDS-PAGE as described above and transferred to nitrocellulose membranes. Membranes were then blocked and immunoblotted with antibodies overnight at 4° C. After washing three times in PBS-Tween the membranes were then incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies. The results were visualized via a chemiluminescent Pierce ECL Substrate Western blot detection system (Thermo Scientific, USA) and exposure to autoradiography film (Kodak XAR film).
[1530] The primary antibody used for detection was goat anti-mouse IgG (H+L) Antibody (LI-COR, Cat. No. 926-32210).
[1531] The result for anti-VISTA antibody clone V4 ([1] of Example 2.2) is shown in
Example 3: Biophysical Characterisation
[1532] 3.1 Analysis of cell surface antigen-binding by flow cytometry
[1533] Wildtype HEK293T cells (which do not express high levels of VISTA) and cells of HEK293T cells transfected with vector encoding human VISTA (i.e. HEK 293 HER O/E cells) were incubated with 20 μg/ml of anti-VISTA antibody or isotype control antibody at 4° C. for 1 hr. The anti-VISTA antibody clone VSTB112, as described in WO 2015/097536, was included in the analysis as a positive control.
[1534] The cells were washed thrice with FACS buffer (PBS with 5 mM EDTA and 0.5% BSA) and resuspended in FITC-conjugated anti-FC antibody (Invitrogen, USA) for 40 min at 2-8° C. Cells were washed again and resuspended in 200 μL of FACS flow buffer (PBS with 5 mM EDTA) for flow cytometric analysis using MACSQuant 10 (Miltenyi Biotec, Germany). After acquisition, all raw data were analyzed using Flowlogic software. Cells were gated using forward and side scatter profile and Median of Fluorescence Intensity (MFI) value was determined for native and overexpressing cell populations.
[1535] The results are shown in
[1536] In a separate experiment 13D5p ([14] of Example 2.2) was analysed for its ability to bind to cells transfected with vector encoding cynomolgus macaque VISTA or murine VISTA. 13D5p was found to display cross-reactivity with cynomolgus macaque VISTA and murine VISTA.
3.2 Global affinity study using Octet QK384 system
[1537] Bio-Layer Interferometry (BLI) experiments were performed using the Octet QK384 system (ForteBio). anti-Penta-HIS (HIS1K) coated biosensor tips (Pall ForteBio, USA) were used to capture His-tagged human, cynomolgus macaque or murine VISTA (270 nM). All measurements were performed at 25° C. with agitation at 1000 rpm. Kinetic measurements for antigen binding were performed by loading anti-VISTA antibody at different concentrations (indicated in the Figures) for 120 s, followed by a 120 s dissociation time by transferring the biosensors into assay buffer containing wells. Sensograms were referenced for buffer effects and then fitted using the Octet QK384 user software (Pall ForteBio, USA). Kinetic responses were subjected to a global fitting using a one site binding model to obtain values for association (kon), dissociation (koff) rate constants and the equilibrium dissociation constant (KD). Only curves that could be reliably fitted with the software (R.sup.2>0.90) were included in the analysis.
[1538] Representative sensorgrams for analysis of binding by anti-VISTA antibody clone V4 (i.e. [1] of example 2.2) are shown in
[1539] Anti-VISTA antibody clone V4 was found to bind to human and cynomolgus macaque VISTA with an affinity of K.sub.D=<1 μM, and to bind to murine VISTA with an affinity of K.sub.D=113 nM.
3.3 ELISAs for Determining Antibody Specificity
[1540] ELISAs were used to determine the binding specificity of the antibodies. Anti-VISTA antibodies were analysed for binding to human VISTA polypeptide, respective mouse and cynomolgus macaque homologues, as well as human PD-L1 and human HER3 (Sino Biological Inc., China).
[1541] ELISAs were carried out according to standard protocols. Briefly, 96-well plates (Nunc, Denmark) were coated with 1 μg/ml of target protein in phosphate-buffered saline (PBS) for 2 h at 37° C. After blocking for 1 h with 10% BSA in Tris buffer saline (TBS) at room temperature, the test antibody was serially diluted (12 point serial dilution) with the highest concentration being 30 μg/ml and added to the plate, in the. Post 1 h incubation at room temperature, plates were washed three times with TBS containing 0.05% Tween 20 (TBS-T) and were then incubated with a HRP-conjugated anti-mouse IgG antibody (Life Technologies, Inc., USA) for 1 h at room temperature. After washing, plates were developed with colorimetric detection substrate 3,3′,5,5′-tetramethylbenzidine (Turbo-TMB; Pierce, USA) for 15 min at room temperature. The reaction was stopped with 2M H2504, and OD was measured at 450 nM within 30 min.
[1542] The results obtained with anti-VISTA antibody clone V4 ([1] of Example 2.2) are shown in
[1543] The results obtained with anti-VISTA antibody clone 13D5-1 ([15] of Example 2.2) are shown in
[1544] In a further experiment, anti-VISTA antibody clone V4 ([1] of Example 2.2) was analysed by ELISA for ability to bind to human VISTA, PD-1, PD-L1, B7H3, B7H4, B7H6, B7H7 and CTLA4. The results are shown in
3.4 Analysis of thermostability by Differential Scanning Fluorimetry
[1545] Briefly, triplicate reaction mixes of antibodies at 0.2 mg/mL and SYPRO Orange dye (ThermoFisher) were prepared in 25 μL of PBS, transferred to wells of MicroAmp Optical 96-Well Reaction Plates (ThermoFisher), and sealed with MicroAmp Optical Adhesive Film (ThermoFisher). Melting curves were run in a 7500 fast Real-Time PCR system (Applied Biosystems) selecting TAMRA as reporter and ROX as passive reference. The thermal profile included an initial step of 2 min at 25° C. and a final step of 2 min at 99° C., with a ramp rate of 1.2%. The first derivative of the raw data was plotted as a function of temperature to obtain the derivative melting curves. Melting temperatures (Tm) of the antibodies were extracted from the peaks of the derivative curves.
[1546] The first derivative of the raw data obtained for Differential Scanning Fluorimetry analysis of the thermostability of antibody clone V4 IgG1 format (i.e. [1] of Example 2.2) is shown in
Example 4: Functional Characterisation
[1547] 4.1 Interaction between VISTA and VSIG-3
[1548] The inventors investigated whether VSIG-3 behaves as a ligand for VISTA by Bio-Layer Interferometry (BLI) analysis using the Octet QK384 system (ForteBio). Briefly, an anti-human Fc capture biosensor was used to capture Fc-tagged VSIG-3 at concentration 100 nM, and association of captured VSIG-3 with VISTA applied at concentrations starting from 3000 nM followed by 3 serial dilutions were measured, and compared to PBS control.
[1549] Representative sensorgrams are shown in
[1550] The inventors next analysed the ability of anti-VISTA antibodies to inhibit interaction between VISTA and VSIG-3.
[1551] Briefly, 96-well plates (Nunc, Denmark) were coated with 1 μg/ml of untagged or Fc-tagged VSIG-3 (R&D Systems, USA) in 1×PBS for 16 h at 4° C. After blocking for 1 h with 1% BSA in TBS at room temperature, 15 μg/ml of VISTA/human His-tagged fusion protein (Sinobiological Inc, China) was added in the presence or absence of increasing concentrations of anti-VISTA antibody, and incubated for 1 hr at room temperature. Plates were subsequently washed three times with TBS-T and incubated with an HRP-conjugated anti-his secondary antibody for 1 h at room temperature. After washing, plates were developed with colorimetric detection substrate Turbo-TMB (Pierce, USA). The reaction was stopped with 2M H2504, and OD was measured at 450 nM.
[1552] The results obtained for anti-VISTA antibody clones 5M1-A11 and 9M2-C12 ([10] and [13] of Example 2.2) are shown in
[1553] In a further experiment, inhibition by 4M2-C12 IgG1 ([1] of Example 2.2) of interaction between VISTA and VSIG-3 was analysed. Inhibition of VISTA:VSIG-3 interaction by an antibody specific for an irrelevant target antigen and by human IgG1 isotype control were also analysed as control conditions. The results are shown in
[1554] In a further experiment, inhibition by 4M2-C12 IgG1 ([1] of Example 2.2) of interaction between VISTA and VSIG-3 was analysed in an assay in which VISTA-Fc was used as the capture agent. Briefly, wells of 384-well plates were coated with 30 μl of 0.5 μg/ml of VISTA-Fc for 1 h at room temperature. Plates were washed with PBS-T and blocked for 1 h with 1% BSA in TBS at room temperature. Serial dilutions of 4M2-C12 IgG1 or human IgG1 isotype control antibodies were added to plates, together with 0.3 μg/ml of VISG3-His. After 1 h of incubation at room temperature plates were washed five times with PBS-T, and incubated with goat anti-HIS-HRP for 1 h at room temperature. Plates were washed five times with PBS-T and, developed with Turbo-TMB. The reaction was stopped with 2M H2504, and OD was measured at 450 nM.
[1555] The results are shown in
4.2 Interaction between VISTA and PSGL-1
[1556] The inventors next investigated whether PSGL-1 behaves as a ligand for VISTA in a flow cytometry-based assay.
[1557] Briefly, 100,000 HEK293T cells modified to overexpress human VISTA protein (by transfection with a construct encoding human VISTA) were co-incubated with 4M2-012-hIgG1 ([1] of Example 2.2) or an isotype-matched control antibody at concentrations of 20 μg/ml, 40 μg/ml or 80 μg/ml for 15 min at 4° C., in buffer comprising HBSS, 0.5% BSA and 2 mM EDTA pH 6.0. 15 μg/ml of Fc-tagged human PSGL1 (R&D Systems, Cat No: 3345-PS) or the same amount of an Fc-tagged irrelevant antigen was then added to the cells, which were then incubated for a further 45 min at 4° C. Cells were subsequently washed three times with buffer, and then FITC-conjugated anti-PSGL1 antibody (Miltenyi Biotec Cat No: 130-104-706) was added at a dilution factor of (1:11) or Alex488-conjugated anti-Fc antibody was added at a dilution factor of 1:200, and the cells were incubated for 15 min at 4° C. Cells were then washed three times with buffer and analysed by flow cytometry.
[1558] The results are shown in
4.3 Inhibition of VISTA-mediated signalling
[1559] The inventors investigated whether anti-VISTA antibody clone 13D5p could inhibit VISTA-mediated signalling by analysis using a mixed lymphocyte reaction (MLR) assay.
[1560] Briefly, PBMCs were isolated from unrelated donors (to obtain stimulator and effector populations) using Septamate kit (Stemcell Technologies, Canada), according to the manufacturer's instructions. Stimulator cells were treated with 50 μg/mL of mitomycin C (Sigma Aldrich, USA) for 20 minutes at 37° C. and used after 5 washes with 1×PBS. The stimulator population was seeded at 0.5×10.sup.5 cells/well and responder population at 1.0×10.sup.5 cells per well in the presence or absence of increasing concentrations of the test antibody, starting at a highest concentration of 20 μg/ml. After 5 days, the supernatant was harvested and the levels of IL-17, IL-2A and IFN-γ were determined by ELISA following the standard protocol.
[1561] The results are shown in
Example 5: Analysis In Vivo
[1562] For in vivo studies, 4M2-C12 was produced in mouse IgG2a format. The molecule is a heteromer of the heavy chain polypeptide having the sequence shown in SEQ ID NO:248, and the light chain polypeptide having the sequence shown in SEQ ID NO:250. 4M2-C12 mIgG2a was produced by co-expression of nucleic acids encoding the heavy and light chains polypeptides in CHO cells, and was subsequently purified.
TABLE-US-00005 Antigen- biding molecule Polypeptides Antibody [17] 4M2-C12 mIgG2a HC 4M2-C12 mIgG2a (SEQ ID NO: 248) + 4M2-C12 CL (SEQ ID NO: 250)
[1563] 5.1 Pharmacokinetic analysis
[1564] C57BL/6 mice approximately 6-8 weeks old were housed under specific pathogen-free conditions and were treated in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines.
[1565] 600 μg anti-VISTA antibody was administered and blood was obtained from 3 mice by cardiac puncture at baseline (−2 hr), 0.5 hr, 6 hr, 24 hr, 96 hr, 168 hr and 336 hr after administration. Antibody in the serum was quantified be ELISA.
[1566] The parameters for the pharmacokinetic analysis were derived from a non-compartmental model: maximum concentration (C.sub.max), AUC (0-336 hr), AUC (0-infinity), Half-life (t.sub.1/2), Clearance (CL), Volume of distribution at steady state (V.sub.ss).
[1567] The results obtained for anti-VISTA antibody clone V4 ([17] of Example 5) are shown in
5.2 Analysis of efficacy to treat cancer in vivo
[1568] Female BALB/c or C57BL/6 mice approximately 6-8 weeks old were purchased from InVivos (Singapore). Animals were housed under specific pathogen-free conditions and were treated in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines.
[1569] Cell lines used in the studies included LL2 cells (Lewis Lung carcinoma), 4T1 cells (breast cancer), CT26 cells (colon carcinoma), Clone-M3 cells (melanoma) and EL4 cells (T cell leukemia/lymphoma) obtained from ATCC. B16-BL6 cells (melanoma) were obtained from Creative Bioarray. The cell lines were maintained in accordance with the supplier's instructions; LL2 cells, B16-BL6 cells and EL4 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% Pen/Strep, and 4T1 cells and CT26 cells were cultured in RPMI-1640 supplemented with 10% FBS and 1 and 1% Pen/Strep. Clone-M3 cells were grown in F12-K medium supplemented with 2.5% FBS, 15% Horse serum and 1% Pen/Strep. All cells were cultured at 37° C. in a 5% CO.sub.2 incubator.
[1570] Syngeneic tumor models were generated by injecting either LL2 (2×10.sup.5), 4T1 (5×10.sup.5), CT26 (1×10.sup.5-1×10.sup.6), Clone-M3 (5×10.sup.5), EL4 (2×10.sup.5) or B16-BL6 (1×10.sup.5) cells subcutaneously into the right flank of mice.
[1571] 3 days post-implantation anti-VISTA antibodies were administered intraperitoneally every 3 days for a total of 6 doses. Control groups received vehicle treatment at the same dose interval.
[1572] Tumor volume was measured 3 times a week using a digital caliper and calculated using the formula [L×W2/2]. Study End point was considered to have been reached once the tumors of the control arm measured >1.5 cm in length.
5.2.1 CT26 cell model
[1573]
[1574] V4 or anti-PD-L1 antibody were administered at 300 μg per dose every 3 days from day 3. A combination treatment of 300 μg V4+300 μg anti-PD-L1 antibody per dose was also included in the analysis.
[1575] Anti-VISTA antibody clone V4 was found to be highly potent in this model, and capable of inhibiting tumor growth by ˜60%.
[1576] At day 21 tumors were harvested and evaluated for Arg1 RNA expression by RNA-seq analysis, according to the method described in Newman et al. Nat Methods. (2015) 12(5):453-457. The results are shown in
[1577] In another experiment a CT26 cell-line derived syngeneic mouse colon carcinoma model was established by subcutaneous injection of 100,000 CT26 cells into the right flank of Balb/c mice (n=8 mice per treatment group), and mic were treated by administration of 300 μg per dose every 3 days from day 3 of an isotype control antibody, anti-PD-L1 antibody clone 10F.9G2, anti-VISTA antibody clone V4 ([17] of Example 5), anti-TIGIT antibody clone 1G9, anti-LAG-3 antibody clone C9B7W, anti-TIM-3 antibody clone RMT3-23, or combination treatments of 300 μg anti-PD-L1 antibody clone 10F.9G2 with 300 μg of each of the other of antibodies per dose was also included in the analysis.
[1578] The results of the calculated inhibition of tumor growth detected at day 15 are shown in
[1579] The inventors performed a further experiment in which CT26 tumors were established in the same way, and mice were then administered biweekly with 300 μg anti-VISTA antibody clone V4, 200 μg anti-PD-L1 antibody clone 10F.9G2, 300 μg anti-VISTA antibody clone V4+200 μg anti-PD-L1 antibody clone 10F.9G2, or PBS as a control condition. At the end of the experiment tumors were analysed by RNA-Seq to determine the relative numbers of MDSCs, CD8+ T cell and Tregs, according to the method described in Newman et al. Nat Methods. (2015) 12(5):453-457 which is hereby incorporated by reference in its entirety.
[1580] The results are shown in
[1581] In a further experiment, a CT26 cell-line derived syngeneic mouse colon carcinoma model was established in Balb/C mice as described above, and mice were administered from day 3 and every 3 days with: (i) 600 μg anti-VISTA antibody clone 13D5-1, (ii) 200 μg anti-PD-L1 antibody clone 10F.9G2, (iii) 600 μg anti-VISTA antibody clone 13D5-1+200 μg anti-PD-L1 antibody clone 10F.9G2, or (iv) an equal volume of PBS (as a negative control).
[1582] The results are shown in
5.2.2 LL2 cell model
[1583] The LL2 model was established by subcutaneous injection of 200,000 LL2 cells into the right flank of Balb/c mice (n=8 mice per treatment group), and mice were subsequently administered biweekly with 600 μg anti-VISTA antibody clone V4 ([17] of Example 5) or an equal volume of vehicle as a negative control.
[1584] The results of the experiment are shown in
5.2.3 B16-BL6 cell model
[1585] The B16-BL6 model was established by subcutaneous injection of 200,000 B16-BL6 cells into the right flank of C57BL/6 mice (n=8 mice per treatment group), and mice were subsequently administered biweekly (for a total of 6 doses) with 600 μg anti-VISTA antibody clone V4 ([17] of Example 5), 200 μg of anti-PD-1 antibody RMP1-14 (Bio X Cell), 600 μg anti-VISTA antibody clone V4+200 μg anti-PD-1 antibody, or an equal volume of vehicle as a negative control.
[1586] The results of the experiment are shown in
5.2.4 4T1 cell model
[1587] The 4T1 cell-line derived syngeneic mouse mammary carcinoma model was established in Balb/c mice by subcutaneous injection of 250,000 4T1 cells into the right flank.
[1588] Mice were subsequently administered from day 3 and every 3 days (for a total of 6 doses) with either 300 or 600 μg of anti-VISTA antibody clone 13D5-1, isotype control antibody or an equal volume of vehicle as a negative control.
[1589] The results of the experiment are shown in
5.3 Safety pharmacology, toxicology and immunotoxicity
[1590] Anti-VISTA antibody clone V4 and humanized versions V4H1 and V4H2 were analysed in silico for safety and immunogenicity using IMGT DomainGapAlign (Ehrenmann et al., Nucleic Acids Res., 38, D301-307 (2010)) and IEDB deimmunization (Dhanda et al., Immunology. (2018) 153(1):118-132) tools.
[1591] Anti-VISTA antibody clones V4H1 and V4H2 had sufficient homology to human heavy and light chains to be considered humanized (i.e. >85%), had numbers of potential immunogenic peptides few enough to be considered safe, and did not possess any other properties that could cause potential developability issues.
[1592] The inventors also weighed and analysed mice for signs of gross necroscopy during the course of the experiments described in Example 5.2; mice treated with anti-VISTA antibody clone V4 did not display any differences from PBS-treated control mice.
[1593] The inventors further investigated hemotoxicity in an experiment in which mice were injected with a single dose of 900 μg anti-VISTA antibody clone V4 or an equal volume of PBS.
[1594] Blood samples were obtained and analysed for numbers of different types of white blood cells using HM5 Hematology Analyser. The results are shown in
[1595] The mice were also analysed for correlates hepatotoxicity and nephrotoxicity, and the results are shown in
5.4 Treatment of Advanced Solid Tumors
First in Human
[1596] Patients with advanced or metastatic solid tumors with disease progression or treatment intolerance after treatment with standard therapies and with adequate organ function and ECOG status are treated by intravenous injection of anti-VISTA antibody V4 ([1] of Example 2.2), V4H1 ([3] of Example 2.2) or V4H2 ([4] of Example 2.2), at a dose calculated in accordance with safety-adjusted ‘Minimal Anticipated Biological Effect Level’ (MABEL) approach. Patients are monitored for 28 days post-administration.
[1597] The patients are then evaluated according to the Common Terminology Criteria for Adverse Events (CTCAE), to determine the safety and tolerability of the treatment, and to determine the pharmacokinetics of the molecules.
[1598] Treatment with the anti-VISTA antibodies is found to be safe and tolerable.
Dose escalation—monotherapy
[1599] Patients with advanced or metastatic solid tumors with disease progression or treatment intolerance after treatment with standard therapies and with adequate organ function and ECOG status (n=18-24) are treated by intravenous injection of anti-VISTA antibody V4 ([1] of Example 2.2), V4H1 ([3] of Example 2.2) or V4H2 ([4] of Example 2.2), in accordance with a 3+3 model based escalation with overdose control (EWOC) dose escalation.
[1600] The patients are then evaluated according to the Common Terminology Criteria for Adverse Events (CTCAE) to determine the safety and tolerability of the treatment, and the pharmacokinetics of the molecules and efficacy of the treatment is evaluated. The maximum tolerated dose (MTD) and maximum administered dose (MAD) are also determined.
Dose escalation—combination therapy
[1601] Patients with advanced or metastatic solid tumors with disease progression or treatment intolerance after treatment with standard therapies and with adequate organ function and ECOG status (n=9) are treated with anti-VISTA antibody V4 ([1] of Example 2.2), V4H1 ([3] of Example 2.2) or V4H2 ([4] of Example 2.2), in accordance with a 3+3 model based escalation with anti-PD-1 or anti-PD-L1 antibody (3 mg/kg).
[1602] The patients are then evaluated according to the Common Terminology Criteria for Adverse Events (CTCAE) to determine the safety and tolerability of the treatment, and the pharmacokinetics of the molecules and efficacy of the treatment is evaluated.
Dose Expansion
[1603] Treated patients are analysed for overall response rate, expression of tumor markers, circulating tumor cells, progression-free survival, overall survival, safety and tolerability.
[1604] The anti-VISTA antibodies are found to be safe and tolerable, to be able to reduce the number/proportion of cancer cells, reduce tumor cell marker expression, increase progression-free survival and increase overall survival.
5.5 Treatment of Lymphoma
First in Human
[1605] Patients with lymphoma (NHL and HL) who did not benefit from 1 line of chemotherapy, who have not received allogeneic stem cell transplantation and are likely to respond to rituximab (NHL) and nivolumab or pembrolizumab (HL) are treated by intravenous injection of anti-VISTA antibody V4 ([1] of Example 2.2), V4H1 ([3] of Example 2.2) or V4H2 ([4] of Example 2.2), at a dose calculated in accordance with safety-adjusted ‘Minimal Anticipated Biological Effect Level’ (MABEL) approach. Patients are monitored for 28 days post-administration.
[1606] The patients are then evaluated according to the Common Terminology Criteria for Adverse Events (CTCAE), to determine the safety and tolerability of the treatment, and to determine the pharmacokinetics of the molecules.
[1607] Treatment with the anti-VISTA antibodies is found to be safe and tolerable.
Dose Escalation—Monotherapy
[1608] Patients with lymphoma (NHL and HL) who did not benefit from 1 line of chemotherapy, who have not received allogeneic stem cell transplantation and are likely to respond to rituximab (NHL) and nivolumab or pembrolizumab (HL) are treated by intravenous injection of anti-VISTA antibody V4 ([1] of Example 2.2), V4H1 ([3] of Example 2.2) or V4H2 ([4] of Example 2.2), in accordance with a 3+3 model based escalation with overdose control (EWOC) dose escalation.
[1609] The patients are then evaluated according to the Common Terminology Criteria for Adverse Events (CTCAE) to determine the safety and tolerability of the treatment, and the pharmacokinetics of the molecules and efficacy of the treatment is evaluated. The maximum tolerated dose (MTD) and maximum administered dose (MAD) are also determined.
Dose Escalation—Combination Therapy
[1610] Patients with lymphoma (NHL and HL) who did not benefit from 1 line of chemotherapy, who have not received allogeneic stem cell transplantation and are likely to respond to rituximab (NHL) and nivolumab or pembrolizumab (HL) are treated by intravenous injection of anti-VISTA antibody V4 ([1] of Example 2.2), V4H1 ([3] of Example 2.2) or V4H2 ([4] of Example 2.2), in accordance with a 3+3 model based escalation with anti-PD-L1 antibody.
[1611] The patients are then evaluated according to the Common Terminology Criteria for Adverse Events (CTCAE) to determine the safety and tolerability of the treatment, and the pharmacokinetics of the molecules and efficacy of the treatment is evaluated.
Dose Expansion
[1612] Treated patients are analysed for overall response rate, expression of cancer cell markers, circulating cancer cells, progression-free survival, overall survival, safety and tolerability.
[1613] The anti-VISTA antibodies are found to be safe and tolerable, to be able to reduce the number/proportion of cancer cells, reduce tumor cell marker expression, increase progression-free survival and increase overall survival.
Example 6: Production and Characterisation of VISTA-Binding Antibodies Comprising Different Fc Regions
[1614] 6.1 Production and characterisation of VISTA-binding antibodies comprising different Fc regions 4M2-C12 was produced in mouse IgG2a LALA PG format. The molecule is a heteromer of the heavy chain polypeptide having the sequence shown in SEQ ID NO:249, and the light chain polypeptide having the sequence shown in SEQ ID NO:250. The heavy chain sequence comprises leucine (L) to alanine (A) substitutions in the CH2 region, at positions 4 and 5 numbered according to SEQ ID NO:253, and a proline (P) to glycine (G) substitution at position 99 numbered according to SEQ ID NO:253. These substitutions are referred to in the literature as L234A, L235A and P329G, and are described in mouse IgG2a Fc e.g. in Lo et al. J. Biol. Chem (2017) 292(9):3900-3908, which is hereby incorporated by reference in its entirety. 4M2-C12 mIgG2a LALA PG was produced by co-expression of nucleic acids encoding the heavy and light chain polypeptides in CHO cells, and was subsequently purified.
TABLE-US-00006 Antigen- biding molecule Polypeptides Antibody [18] 4M2-C12 mIgG2a LALA PG HC 4M2-C12 mIgG2a (SEQ ID NO: 249) + LALA PG 4M2-C12 CL (SEQ ID NO: 250)
[1615] 4M2-C12 was also produced in mouse IgG2a NQ format. The molecule is a heteromer of the heavy chain polypeptide having the sequence shown in SEQ ID NO:258, and the light chain polypeptide having the sequence shown in SEQ ID NO:250. The heavy chain sequence comprises an asparagine (N) to glutamine (Q) substitution in the CH2 region, at position 67 according to SEQ ID NO:253. This substitution is referred to in the literature as N297Q, and is described in mouse IgG2a Fc e.g. in Lo et al. J. Biol. Chem (2017) 292(9):3900-3908. 4M2-C12 mIgG2a NQ was produced by co-expression of nucleic acids encoding the heavy and light chain polypeptides in CHO cells, and was subsequently purified.
TABLE-US-00007 Antigen- biding molecule Polypeptides Antibody [19] 4M2-C12 mIgG2a NQ HC 4M2-C12 mIgG2a NQ (SEQ ID NO: 258) + 4M2-C12 CL (SEQ ID NO: 250)
[1616] 4M2-C12 was produced in mouse IgG1 format. The molecule is a heteromer of the heavy chain polypeptide having the sequence shown in SEQ ID NO:266, and the light chain polypeptide having the sequence shown in SEQ ID NO:250. 4M2-C12 mIgG1 was produced by co-expression of nucleic acids encoding the heavy and light chains polypeptides in CHO cells, and was subsequently purified.
TABLE-US-00008 Antigen- biding molecule Polypeptides Antibody [20] 4M2-C12 mIgG1 HC 4M2-C12 mIgG1 (SEQ ID NO: 266) + 4M2-C12 CL (SEQ ID NO: 250)
[1617] 6.2 Analysis of binding of VISTA-binding antibodies comprising different Fc regions to Fc receptors Binding of 4M2-C12 in different antibody formats to human, cynomolgus and murine VISTA protein was assessed via ELISA, and binding to mouse Fcγ receptors and mouse FcRn was assessed by BLI using a Pall Forte Bio Octet QK 384 system.
[1618] Histidine-tagged mFcγRIV (50036-M08H), mFcγRIII (50326-M08H), mFcγRIIb (50030-M08H), and mFcRn (CT009-H08H) were obtained from Sino Biological. Anti-Penta-HIS (HIS1K) biosensors were purchased from Forte Bio (18-5120).
[1619] For the kinetic experiment, anti-Penta-HIS biosensors were incubated for 60 sec in PBS buffer (pH 7.2) to obtain the first baseline, and were subsequently loaded for 120 sec with 200 nM mFcγRIV (orthologue of hFcγRIIIa), 160 nM mFcγRIII (orthologue of hFcγRIIa), 75 nM mFcγRIIb (orthologue of hFcγRIIb) or 120 nM mFcRn in PBS (pH 7.2). After loading, biosensors were incubated for 60 sec in PBS buffer (pH 7.2 for Fcγ receptors and pH 5.8 for FcRn) to obtain the second baseline, and for 60 sec with a 6 point 2-fold dilution series of the test antibodies (2000 nM-62.5 nM for mFcγ receptor binding, and 500 nM-15.6 nM for FcRn binding) in PBS (pH 7.2 for Fcγ receptors and pH 5.8 for FcRn) to obtain the association curves. Finally, the biosensors were incubated for 120 sec in PBS (pH 7.2 for mFcγR and pH 5.8 for mFcRn) to obtain the dissociation curves. Kinetic and affinity constants were calculated by global fitting of the association and dissociation data to a 1:1 binding model.
[1620] The results for analysis of binding of 4M2-012-mIgG1 to different mouse Fcγ receptors and mouse FcRn are shown in
[1621] The results for analysis of binding of 4M2-012-mIgG2a to different mouse Fcγ receptors and mouse FcRn are shown in
[1622] The results for analysis of binding of 4M2-012-mIgG2a LALA PG to different mouse Fcγ receptors and mouse FcRn are shown in
[1623] The results for analysis of binding of 4M2-C12-mIgG2a NQ to different mouse Fcγ receptors and mouse FcRn are shown in
[1624] The K.sub.on, K.sub.dis and K.sub.D values for binding to different Fc receptors determined for 4M2-C12-mIgG1, 4M2-C12-mIgG2a, 4M2-C12-mIgG2a LALA PG and 4M2-C12-mIgG2a NQ are summarised in the tables of
Example 7: Analysis In Vivo of VISTA-Binding Antibodies Comprising Different Fc Regions
[1625] 4M2-012-mIgG2a and 4M2-012-mIgG2a LALA PG (see Example 6.1) were evaluated for efficacy to treat cancer in vivo in a syngeneic EL4 T-cell leukemia/lymphoma model.
[1626] EL4 cells cultured in DMEM supplemented with 10% Horse serum (FBS) and 1% Pen/Strep. Cells were cultured at 37° C. in a 5% 002 incubator.
[1627] C57BL/6 mice, approximately 6 weeks old were obtained from InVivos (Singapore). Animals were housed under specific pathogen-free conditions and were treated in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines. C57BL/6 mice were inoculated with 2×10.sup.5 EL4 T-cell leukemia/lymphoma cells on the right flank. Post tumor implantation, when tumors reached 350 to 400 mm.sup.3 in size, mice were randomized to the following treatment groups: a) vehicle control (PBS), b) 4M2-C12 mIgG2a ([17] of Example 5), or c) 4M2-C12 mIgG2a-LALA PG ([18] of Example 6), at a dose of 25 mg/kg. The treatments were administered intraperitoneally every 3 days for a total of 5 doses.
[1628] Tumor volume was measured 3 times a week using a digital caliper, and calculated using the formula [L×W2/2]. Study End point was considered to have been reached once the tumors of the vehicle control treatment group measured >1.5 cm in length.
[1629] The results are shown in
[1630] Thus 4M2-C12 was found to display potent inhibition of tumor growth in both IgG2a and IgG2a LALA PG formats.
[1631] Treatment of highly established EL4 tumor-bearing mice with anti-VISTA antibody 4M2-C12 was found to be very effective as a monotherapy, and resulted in tumor clearance.
[1632] The biological activity of 4M2-C12 was found not to be dependent on engagement of murine Fcγ receptors, strongly suggesting that 4M2-C12 exerts its biological activity through inhibition of VISTA-mediated signalling.
Example 8: Analysis of the Epitope of VISTA Bound by the Antibodies
[1633] Anti-VISTA antibodies were evaluated to determine whether they compete with one another for binding to VISTA.
[1634] VSTB112 has previously been suggested to bind VISTA in several regions. The major epitopes have been proposed to correspond to positions 59 to 68 and positions 86 to 97 of SEQ ID NO:1 (i.e. SEQ ID NOs:271 and 272). The minor epitopes have been proposed to correspond to positions 71 to 84 and positions 150 to 166 of SEQ ID NO:1 (i.e. SEQ ID NOs:273 and 274); see e.g. WO 2017/137830 A1, e.g. at paragraph [0302]. VSTB112 is described e.g. in WO 2015/097536 A2, which is hereby incorporated by reference in its entirety.
[1635] IGN175A is thought to bind to VISTA within the first 32 amino acids of the mature protein (i.e. within positions 33 to 64 of SEQ ID NO:1 (i.e. SEQ ID NO:275)). IGN175A is described e.g. in WO 2014/197849 A2, which is hereby incorporated by reference in its entirety.
[1636] Epitope binning experiments were performed by BLI using the Octet QK384 system (ForteBio). Briefly, human VISTA-His recombinant protein in PBS (4.7 μg/ml) was immobilized to Anti-Penta His sensor (HIS1K, ForteBio), for 5 mins. Baseline signals in PBS were measured for 30s before loading of 400 nM saturating antibody in PBS for 10 mins, and at a shake speed of 1000 rpm, followed by a 120 s dissociation step using PBS. Biosensors were subsequently treated with 300 nM competing antibody in
[1637] PBS for 5 mins, at a shake speed of 1000 rpm, followed by a 120 s dissociation step using PBS.
[1638] The following antigen-binding molecules were analysed in the experiment: [1639] 4M2-C12 (V4) in IgG1 format ([1] of Example 2.2) [1640] A humanised and affinity-matured variant of 4M2-C12 (V4-C1) in IgG1 format ([21] of Example 13) [1641] IGN175A IgG1 (comprising IGN175A HC (SEQ ID NO: 267)+IGN175A LC (SEQ ID NO: 268)) [1642] VSTB112 IgG1 (comprising VSTB112 HC (SEQ ID NO: 269)+VSTB112 LC (SEQ ID NO: 270))
[1643] The following antigen/saturating antibody/competing antibody combinations were investigated:
TABLE-US-00009 Antigen Saturating Antibody Competing Antibody human VISTA V4-C1 IgG1 IGN175A IgG1 human VISTA V4-C1 IgG1 V4-C1 IgG1 human VISTA V4-C1 IgG1 VSTB112 IgG1 human VISTA VSTB112 IgG1 IGN175A IgG1 human VISTA IGN175A IgG1 V4-C1 IgG1 human VISTA VSTB112 IgG1 V4-C1 IgG1 human VISTA IGN175A IgG1 IGN175A IgG1 None (PBS) V4-C1 IgG1 IGN175A IgG1
[1644] The results are shown in
[1645] V4 and V4-C1 IgG1 were found not to compete with IGN175A for binding to VISTA. VSTB112 was found to partially compete with V4, V4-C1 and IGN175A for binding to VISTA. Changes in response (in nm) upon addition of the competing antibody are shown below.
TABLE-US-00010 Saturating Competing Antibody Antibody IGN175A V4-C1 IgG1 VSTB112 IGN175A 0.0454 1.4362 — V4-C1 IgG1 1.0661 −0.0158 −0.0124 VSTB112 0.1392 0.1579 —
[1646] The results indicate that 4M2-C12 and IGN175A bind to topically distant regions of VISTA, and that VSTB112 binds to VISTA in regions which are proximal to 4M2-C12 and IGN175A.
[1647] The fact that V1-C1 and IGN175A do not compete for binding to VISTA taken together with the observation that VSTB112 competes with IGN175A for binding to VISTA indicates that antibodies comprising the CDRs of 4M2-C12 bind to an epitope of VISTA which is non-identical to the epitope of VISTA bound by IGN175A, and which is also non-identical to the epitope of VISTA bound by VSTB112.
[1648] From analysis of the sequence for VISTA, the immunogen used to raise 4M2-C12 and species cross-reactivity data, the inventors concluded that 4M2-C12 and derivatives thereof bind to the sequence shown in SEQ ID NO:322 (which corresponds to positions 76 to 81 of SEQ ID NO:1).
Example 9: Analysis of the Ability of VISTA-Binding Antibodies to Rescue VISTA-Mediated Inhibition of T Cell Proliferation
[1649] The ability of anti-VISTA antibodies to rescue the inhibitory effects of VISTA-mediated signalling was analysed in an in vitro assay.
[1650] Briefly, 96-well plates were coated with anti-CD3 and VISTA-Ig or control-Ig at concentration ratios of either 1:1 (2.5 μg/ml anti-CD3:2.5 μg/ml of VISTA/control Ig) or 2:1 (2.5 μg/ml anti-CD3: 1.25 μg/ml VISTA/control Ig). Irrelevant antigen-Ig was used as control condition. Plates were incubated overnight at 4° C.
[1651] PBMCs were purified from freshly collected blood samples and further enriched for T cells using human Pan T Cell Isolated Kit (Miltenyi Biotec). The enriched T cell populations were then labelled with CFSE.
[1652] Wells were washed three times with PBS, and 100,000 CFSE-labelled T cells were added to each well, in complete RPMI 1640 medium supplemented with 10% FBS, in the presence of 4M2-C12 IgG1 ([1] of Example 2.2) at a final concentration of 20 μg/ml or 50 μg/ml, or in the presence of VSTB 1 12 at a final concentration of 20 μg/ml, or in the absence of added antibody.
[1653] After 5 days, cells were harvested, labelled with fluorescently conjugated anti-CD4 and anti-CD8 antibodies, and analysed by flow cytometry using a Macsquant Analyzer 10.
[1654] The results of the experiments are shown in
[1655] Importantly, 4M2-C12 was found to be more effective at restoring proliferation of T cells than VSTB112.
Example 10: Analysis of the Ability of VISTA-Binding Antibodies to Promote Production of IL-6 by THP1 Cells in Response to LPS
[1656] The ability of anti-VISTA antibodies to promote production of IL-6 by THP-1 cells in response to LPS stimulation was analysed in an in vitro assay.
[1657] Briefly, undifferentiated THP1 cells were seeded in 96 well plates in duplicate (100,000 cells/well), in RPMI media without FBS or pen/strep. Cells subsequently treated with LPS (final concentration of 100 μg/ml) and MnCl.sub.2(100 μM), in the presence of different concentrations of 4M2-C12 IgG1 ([1] of Example 2.2) ranging from 2000 μg/ml to 7.8 μg/ml, or different concentrations of VSTB112 ranging from 1000 μg/ml to 7.8 μg/ml. After 3 days, the cell culture supernatant was collected and analysed by ELISA to determine the level of IL-6, using the IL-6 Human ELISA Kit (Invitrogen).
[1658] The results are shown in
Example 11: Analysis of the Ability of VISTA-Binding Antibodies to Promote Production of IL-6 In Vivo
[1659] IL-6 production in response to treatment with 4M2-C12 was investigated in vivo.
[1660] Briefly, C57BL/6 mice (n=3) were administered with a single 600 μg dose of 4M2-C12 mIgG2a ([17] of Example 5), and blood samples were harvested from mice at 2 hr before administration, and 0.5 hr, 6 hr, 24 hr, 96 hr, 168 hr and 336 hr post-administration.
[1661] The serum was analysed for IL-6 content using the Mouse IL-6 ELISA Kit (Abcam, ab100712).
[1662] The results are shown in
Example 12: Analysis In Vivo of VISTA-Binding Antibodies Alone or in Combination with Anti-PD-1/PD-L1 Antibody
12.1 CT26 Cell Model
[1663] A syngeneic model of T cell leukemia/lymphoma was generated by injecting 1×10.sup.5CT26 cells subcutaneously into the right flank of Balb/c mice.
[1664] Mice (7 per treatment group) were administered intraperitoneally every 3 days for a total of 7 doses with: [1665] 600 μg of 4M2-C12 IgG2a ([17] of Example 5) [1666] 200 μg of anti-PD-1 antibody (clone RMP1-14 (Bioxcell)) [1667] 600 μg of 4M2-C12 IgG2a+200 μg of anti-PD-1 antibody [1668] PBS only
[1669] Tumor volume was measured 3 times a week using a digital caliper and calculated using the formula [L×W2/2]. Study End point was considered to have been reached once the tumors of the control arm measured >1.5 cm in length.
[1670] The results are shown in
[1671] Immunoprofiling of the tumor-infiltrating CD45+ cells was undertaken. Briefly, at day 22 of the experiment tumors were harvested, processed into single cell suspensions and stained with antibodies specific for immune cell surface proteins (CD45, CD4, CD8, CD25, CD11b, Ly6G, and Ly6C).
[1672] Samples were analysed by flow cytometry, and were classified into the following immune cell subsets based on their staining for the different immune cell surface proteins as follows: [1673] CD4 cells: CD45.sup.+CD4.sup.+; [1674] CD8 T cells: CD45.sup.+CD8.sup.+; [1675] Treg cells: CD45.sup.+CD4.sup.+CD25.sup.+; [1676] Granulocytic MDSC (g-MDSC): CD45.sup.+CD11b.sup.+Ly6G.sup.+Ly6G.sup.+C.sup.lo/− [1677] Monocytic MDSC (m-MDSC): CD45.sup.+CD11b.sup.+Ly6G.sup.−Ly6C.sup.hi/+
[1678] The percentage of tumor-infiltrating CD45+ cells having the indicated phenotypes are summarised below:
TABLE-US-00011 CD4 CD8 Treatment Group cells cells Treg g-MDSC m-MDSC PBS 1.03% 5.99% 0.09% 33.08% 9.8% 4M2-C12 IgG2a 1.66% 6.51% 0.11% 26.4% 8.34% anti-PD-1 1.05% 7.21% 0.14% 46.2% 15.25% antibody 4M2-C12 IgG2a + 1.55% 9.98% 0.22% 15.66% 14.04% anti-PD-1 antibody
[1679] The percentage of tumor-infiltrating CD45+ cells which were g-MDSC is shown in
[1680] Blood was obtained from mice at day 18, and serum was analysed for the levels of various different cytokines by analysis using the MACSPIex cytokine 10 Kit for mouse (Miltenyi Biotec).
[1681] The results are shown in
12.2 B16-BL6 Cell Model
[1682]
[1683] Immunoprofiling of the tumor-infiltrating CD45+ cells was undertaken. Briefly, at day 18 of the experiment tumors were harvested, processed into single cell suspensions, stained with antibodies and specific for immune cell surface, analysed by flow cytometry and cells were classified into immune cell subsets as described in Example 12.1 above.
[1684] The percentage of tumor-infiltrating CD45+ cells having the indicated phenotypes are summarised below:
TABLE-US-00012 CD4 CD8 Treatment Group cells cells Treg g-MDSC m-MDSC PBS 2.72% 8.79% 1.42% 2.06% 6.68% 4M2-C12 IgG2a 2.84% 10.7% 1.58% 1.65% 13.94% anti-PD-1 0.95% 3.43% 0.51% 36.65% 14.12% antibody 4M2-C12 IgG2a + 3.58% 10.6% 0.82% 5.45% 14.44% anti-PD-1 antibody
[1685] The percentage of tumor-infiltrating CD45+ cells which were g-MDSC is shown in
12.3 EL4 Cell Model
[1686] A syngeneic model of T cell leukemia/lymphoma was established by injecting 2×10.sup.5 EL4 cells subcutaneously into the right flank of C57BL/6 mice.
[1687] Mice (7 per treatment group) were administered intraperitoneally every 3 days for a total of 5 doses with: [1688] 600 μg of 4M2-C12 IgG2a ([17] of Example 5) [1689] 200 μg of anti-PD-1 antibody (clone RMP1-14 (Bioxcell)) [1690] 600 μg of 4M2-C12 IgG2a+200 μg of anti-PD-1 antibody [1691] PBS only
[1692] Tumor volume was measured 3 times a week using a digital caliper and calculated using the formula [L×W2/2]. Study End point was considered to have been reached once the tumors of the control arm measured >1.5 cm in length.
[1693] The results are shown in
[1694] Immunoprofiling of the tumor-infiltrating CD45+ cells was undertaken. Briefly, at day 16 of the experiment tumors were harvested, processed into single cell suspensions, stained with antibodies and specific for immune cell surface, analysed by flow cytometry and cells were classified into immune cell subsets as described in Example 12.1 above.
[1695] The percentage of tumor-infiltrating CD45+ cells having the indicated phenotypes are summarised below:
TABLE-US-00013 CD4 CD8 Treatment Group cells cells Treg g-MDSC m-MDSC PBS 3.71% 0.55% 0.18% 19.41% 0.88% 4M2-C12 IgG2a 7.4% 1.84% 0.19% 14.51% 0.94% anti-PD-1 4.35% 3.04% 0.09% 20.91% 0.81% antibody 4M2-C12 IgG2a + 6.53% 2.18% 0.35% 10.16% 0.33% anti-PD-1 antibody
[1696] The percentage of tumor-infiltrating CD45+ cells which were g-MDSC is shown in
12.4 Conclusions
[1697] Inhibition of PD-1/PD-L1 signalling increases the proportion of g-MDSCs amongst the tumor-infiltrating CD45+ cells in the CT26, B16-BL6 and EL4 models, whereas treatment with 4M2-C12 suppresses g-MDSC expansion.
Example 13: Further Characterisation of VISTA-Binding Antibodies
[1698] Further VISTA-binding antigen-binding molecules were produced:
TABLE-US-00014 Antigen- biding molecule Polypeptides Antibody [21] V4-C1 VH-CH1-CH2-CH3 anti-VISTA clone (SEQ ID NO: 311) + V4-C1 IgG1 V4-C1 VL-Cκ (SEQ ID NO: 312) [22] V4-C9 VH-CH1-CH2-CH3 anti-VISTA clone (SEQ ID NO: 313) + V4-C9 IgG1 V4-C9 VL-Cκ (SEQ ID NO: 314) [23] V4-C24/C26/C27/C28/C30/C31 VH-CH1- anti-VISTA clone CH2-CH3 (SEQ ID NO: 315) + V4-C24 IgG1 V4-C24 VL-Cκ (SEQ ID NO: 316) [24] V4-C24/C26/C27/C28/C30/C31 VH-CH1- anti-VISTA clone CH2-CH3 (SEQ ID NO: 315) + V4-C26 IgG1 V4-C26 VL-Cκ (SEQ ID NO: 317) [25] V4-C24/C26/C27/C28/C30/C31 VH-CH1- anti-VISTA clone CH2-CH3 (SEQ ID NO: 315) + V4-C27 IgG1 V4-C27 VL-Cκ (SEQ ID NO: 318) [26] V4-C24/C26/C27/C28/C30/C31 VH-CH1- anti-VISTA clone CH2-CH3 (SEQ ID NO: 315) + V4-C28 IgG1 V4-C28 VL-Cκ (SEQ ID NO: 319) [27] V4-C24/C26/C27/C28/C30/C31 VH-CH1- anti-VISTA clone CH2-CH3 (SEQ ID NO: 315) + V4-C30 IgG1 V4-C30 VL-Cκ (SEQ ID NO: 320) [28] V4-C24/C26/C27/C28/C30/C31 VH-CH1- anti-VISTA clone CH2-CH3 (SEQ ID NO: 315) + V4-C31 IgG1 V4-C31 VL-Cκ (SEQ ID NO: 321)
[1699] V4-C1, V4-C9, V4-C24, V4-026, V4-C27, V4-C28, V4-C30 and V4-C31 were analysed in silico for safety and immunogenicity using IMGT DomainGapAlign (Ehrenmann et al., Nucleic Acids Res., 38, D301-307 (2010)) and IEDB deimmunization (Dhanda et al., Immunology. (2018) 153(1):118-132) tools.
[1700] V4-C1, V4-C9, V4-C24, V4-026, V4-C27, V4-C28, V4-C30 and V4-C31 had sufficient homology to human heavy and light chains to be considered humanized (i.e. >85%), had numbers of potentially immunogenic peptides few enough to be considered safe (see
13.1 Analysis of Binding Affinity by BLI
[1701] Binding of V4-C1, V4-09, V4-024, V4-C26, V4-C27, V4-C28, V4-C30 and V4-C31 (i.e. [21] to [28]) to human and mouse VISTA proteins and human PD-L1 was assessed by BLI using a Pall ForteBio Octet QK 384 system.
[1702] Briefly, anti-Penta-HIS biosensors were incubated for 60 sec in PBS buffer (pH 7.2) to obtain the first baseline, and were subsequently loaded for 120 sec with 180 nM hVISTA, 180 nM mVISTA or 250 nM hPD-L1 in PBS (pH 7.2). After loading, biosensors were incubated for 60 sec in PBS buffer pH 7.2 to obtain the second baseline, and for 120 sec or 900 sec with a 6 point, 2 fold dilution series of the test antibodies (500 nM-15.6 nM) in PBS pH 7.2 to obtain the association curves. Finally, the biosensors were incubated for 120 sec in PBS pH 7.2 to obtain the dissociation curves. Kinetic and affinity constants were calculated by global fitting of the association and dissociation data to a 1:1 binding model.
[1703] None of V4-C1, V4-09, V4-C24, V4-C26, V4-C27, V4-C28, V4-C30 or V4-C31 displayed significant binding to human PD-L1 (
[1704] The kinetic and thermodynamic constants calculated for binding of V4-C1, V4-C9, V4-C24, V4-C26, V4-C27, V4-C28, V4-C30 and V4-C31 to human VISTA and mouse VISTA in this experiment are shown in
[1705] Binding to mouse VISTA protein by V4-C1, V4-C9, V4-C24, V4-C26, V4-C27, V4-028, V4-C30 and V4-C31 was analysed in a separate experiment, which included evaluation of VSTB112 IgG1 (comprising VSTB112 HC (SEQ ID NO: 269)+VSTB112 LC (SEQ ID NO: 270)).
[1706] VSTB112 did not display significant binding to mouse VISTA protein (
[1707] In a further experiment, binding of V4-C1, V4-09, V4-C24, V4-C26, V4-C27 and VSTB112 IgG1 to human VISTA and mouse VISTA was analysed, and the calculated kinetic and thermodynamic constants are shown in
[1708] In another experiment, binding of V4 ([1] of Example 2.2) and VSTB112 IgG1 (comprising VSTB112 HC (SEQ ID NO: 269)+VSTB112 LC (SEQ ID NO: 270)) to human VISTA, mouse VISTA and human CD47 was analysed. Anti-Penta-HIS biosensors were incubated for 60 sec in PBS buffer (pH 7.2) to obtain the first baseline, and were subsequently loaded for 120 sec with 180 nM hVISTA, 180 nM mVISTA or 300 nM hCD47 in PBS (pH 7.2). After loading, biosensors were incubated for 60 sec in PBS buffer pH 7.2 to obtain the second baseline, and for 120 sec with a dilution series of the test antibodies (1500 nM-46.9 nM) in PBS pH 7.2 to obtain the association curves. Finally, the biosensors were incubated for 120 sec in PBS pH 7.2 to obtain the dissociation curves. Kinetic and affinity constants were calculated by global fitting of the association and dissociation data to a 1:1 binding model.
[1709] Neither V4 nor VSTB112 displayed binding to human CD47. VSTB112 did not display significant binding to mouse VISTA protein, whereas V4 did. The calculated kinetic and thermodynamic constants are shown in
13.2 Analysis of Binding Affinity by ELISA
[1710] ELISAs were used to evaluate binding of different antibodies to human VISTA and mouse VISTA. The ELISAs were performed as described in Example 3.3 above.
[1711] The following antibodies were analysed in the experiments: [1712] 4M2-C12 IgG1 ([1] of Example 2.2; referred to as “V4pr” in the Figures) [1713] V4-C1 IgG1 ([21] of Example 13) [1714] V4-C9 IgG1 ([22] of Example 13) [1715] V4-C24 IgG1 ([23] of Example 13) [1716] V4-C26 IgG1 ([24] of Example 13) [1717] V4-C27 IgG1 ([25] of Example 13) [1718] V4-C28 IgG1 ([26] of Example 13) [1719] V4-C30 IgG1 ([27] of Example 13) [1720] V4-C31 IgG1 ([28] of Example 13) [1721] VSTB112 IgG1 (comprising VSTB112 HC (SEQ ID NO: 269)+VSTB112 LC (SEQ ID NO: 270)) [1722] Atezolizumab [1723] Human IgG1 Isotype control
[1724] The results obtained are shown in
[1725] A further experiment was performed in which binding of V4-C1, V4-C9, V4-024, V4-C26, V4-027, V4-C28, V4-C30, V4-C31, VSTB112 and isotype control antibody to human VISTA or mouse VISTA was analysed. The results are shown in
[1726] A further experiment was performed in which binding of V4-C1, V4-C9, V4-024, V4-C26, V4-C27, VSTB112 and isotype control antibody to human VISTA or mouse VISTA was analysed. The results are shown in
[1727] A further experiment was performed in which binding of V4, V4-C24, V4-026, V4-C27, V4-C28, V4-C30 and V4-C31 and isotype control antibody to human VISTA, PD-L1, B7H3, B7H4, B7H6, B7H7, PD-1 and CTLA-4 was analysed. The results are shown in
[1728] In a further experiment, V4 (referred to in
[1729] V4 and all of the V4-derived clones V4-C24, V4-C26, V4-C27, V4-C28, V4-C30 and V4-C31 were found to bind to human VISTA, mouse VISTA, rat VISTA and cyno VISTA.
13.3 Analysis of Binding to VISTA-Expressing Cells by Flow Cytometry
[1730] Anti-VISTA antibodies were analysed for their ability to bind to VISTA-expressing cells essentially as described in Example 3.1 above.
[1731] Briefly, transfected cells, or HEK293 cells transfected with vector encoding human VISTA or mouse VISTA were incubated with 1 μg/ml of anti-VISTA antibody or isotype control antibody at 4° C. for 1 hr. Cells were then washed, and incubated with 10 μg/ml FITC-conjugated anti-human Fc antibody at 4° C. for 1 hr. Cells were washed again, and then analysed by flow cytometry.
[1732] The following antibodies were analysed in the experiments: [1733] 4M2-C12 IgG1 ([1] of Example 2.2; referred to as “V4P” in the Figures) [1734] V4-C24 IgG1 ([23] of Example 13) [1735] V4-C26 IgG1 ([24] of Example 13) [1736] V4-C27 IgG1 ([25] of Example 13) [1737] V4-C28 IgG1 ([26] of Example 13) [1738] V4-C30 IgG1 ([27] of Example 13) [1739] V4-C31 IgG1 ([28] of Example 13) [1740] VSTB112 IgG1 (comprising VSTB112 HC (SEQ ID NO: 269)+VSTB112 LC (SEQ ID NO: 270)) [1741] Human IgG1 Isotype control
[1742] The results are shown in
13.4 Analysis of Thermostability by Differential Scanning Fluorimetry
[1743] Thermostability of different antibodies was evaluated by Differential Scanning Fluorimetry analysis, as described in Example 3.4 above.
[1744] The first derivative of the raw data obtained for Differential Scanning Fluorimetry analysis of the thermostability of V4-C1, V4-C9, V4-C24, V4-C26, V4-C27, V4-C28, V4-C30, V4-C31 (i.e. [21] to [28]) and VSTB112 (in triplicate) is shown in
[1745] V4-C1, V4-C9, V4-C24, V4-026, V4-C27, V4-C28, V4-C30 and V4-C31 were found to have a higher melting temperature (Tm) for the Fab region as compared to V4 (67.5° C.), and thus improved thermal stability.
Example 14: Use of VISTA-Binding Antibodies in Immunohistochemistry
[1746] Anti-VISTA antibody 4M2-C12 mIgG2a ([17] of Example 5) was evaluated for its ability to be used in immunohistochemistry for the detection of human VISTA protein.
[1747] Processing of sections was performed using Bond reagents (Leica Biosystems). Commercial paraffin sections from normal human spleen or normal human ovary were de-paraffinized in Bond Dewax solution, and rehydrated using. Sections were then subjected to the following treatments with 4-5 rinses of 1× Bond Wash between steps: (i) antigen exposure by treatment with Bond Epitope Retrieval Solution for 40 min at 100° C., (ii) endogenous peroxidase blocking by treatment with 3.5% (v/v) H.sub.2O.sub.2 for 15 min at room temperature, (iii) blocking by treatment with 10% goat serum for 30 min at room temperature, (iv) incubation with 4M2-C12 mIgG2a at 1:50 dilution of a 9.37 mg/mL solution overnight at 4° C., (v) incubation with HRP-polymer conjugated goat anti-mouse antibody for 5 min at room temperature, and (vi) development with Bond Mixed DAB Refine for 7 min at room temperature, followed by rinsing with deionised water to stop the reaction.
[1748] Sections were counterstained with haematoxylin for 5 min at room temperature and rinsed with deionised water and 1× Bond Wash solution, and were then dehydrated, mounted in synthetic mounting media and scanned with high resolution.
[1749] The results are shown in
Example 15: Further Analysis of the Ability of VISTA-Binding Antibodies to Rescue VISTA-Mediated Inhibition of T Cell Proliferation and Production of Proinflammatory Cytokines
[1750] Anti-VISTA antibodies were characterised for the ability to release T cells from VISTA-mediated suppression.
[1751] 96-well plates were coated with anti-CD3 at concentration of 2.5 μg/ml and incubated overnight at 4° C. PBMCs were isolated from blood samples, T cells were enriched from the PBMCs and labelled with CSFE as above, and the CFSE-labelled T cell were then co-cultured at a ratio of 2:1 with HEK293-6E cells transfected with a construct encoding human VISTA, in RPMI 1640 medium supplemented with 2% FBS.
[1752] Cells were then treated with 4M2-C12-hIgG1 ([1] of Example 2.2) or VSTB112 at concentrations of 0 μg/ml (control), 20 μg/ml or 50 μg/ml.
[1753] After 5 days, cells were harvested and analysed by flow cytometry to determine cell proliferation by CSFE dilution profile. Cell culture supernatants were also harvested, and INFγ and TNFa levels was analysed by ELISA.
[1754] The results are shown in
[1755] In further experiments, undifferentiated THP1 cells were seeded in wells of 96 well plates in duplicate, in RPMI media without FBS or pen/strep (100,000 cells/well), and cells were stimulated with LPS (100 μg/ml) in the presence of serially diluted concentrations of 4M2-C12-hIgG1 ([1] of Example 2.2) or VSTB112, at concentrations ranging from 2000 μg/ml to 7.8 μg/ml.
[1756] After 24 h cell culture supernatant was collected and analyzed by ELISA for IL-6 and TNFa. Cells were also fixed and permeabilized, and analyzed for the presence of VISTA via flow cytometry.
[1757] The results are shown in
[1758] In a further experiment, undifferentiated THP1 cells were seeded in wells of 96 well plates in duplicate, in RPMI media without FBS or pen/strep (100,000 cells/well), and cells were stimulated with LPS (100 μg/ml) and MnCl.sub.2 (100 μM) in the presence of 4M2-012-hIgG1 ([1] of Example 2.2) or 4M2-012-hIgG4 ([29] shown below), at concentrations ranging from 2000 μg/ml to 7.8 μg/ml. After 24 h cell culture supernatant was collected and analyzed by ELISA for IL-6.
TABLE-US-00015 Antigen- biding molecule Polypeptides Antibody [29] 4M2-C12 VH-CH1-CH2-CH3 IgG4 anti-VISTA clone (SEQ ID NO: 330) + 4M2-C12 IgG4 4M2-C12 VL-Cκ (SEQ ID NO: 213)
[1759] The results are shown in
Example 16: Further Analysis of Pharmacology, Toxicology and Immunotoxicity
[1760] In an acute dose study, rats were administered with a single dose of 10 mg/kg, 25 mg/kg, 100 mg/kg or 250 mg/kg of 4M2-012-hIgG1 ([1] of Example 2.2) or 4M2-012-hIgG4 ([29] of Example 15).
[1761] Blood was obtained from the rats at baseline (−2 hr), 0.5 hr, 6 hr, 24 hr, 96 hr, 168 hr and 336 hr after administration. Antibody in the serum was quantified be ELISA.
[1762] The parameters for the pharmacokinetic analysis were derived from a non-compartmental model: maximum concentration (C.sub.max), AUC (0-336 hr), AUC (0-infinity), Half-life (t.sub.1/2), Clearance (CL), Volume of distribution at steady state (V.sub.ss).
[1763] The results are shown in
[1764] In separate experiments, BALB/C mice were administered with a single dose of 50 mg/kg 4M2-C12-hIgG1 ([1] of Example 2.2) or an equal volume of PBS. Blood samples were obtained after 96 hours, and analysed for numbers of different types of white blood cells using HM5 Hematology Analyser. Blood samples were also analysed for correlates hepatotoxicity and nephrotoxicity.
[1765] Representative results are shown in the tables of
[1766] In further experiments, Sprague Dawley rats were administered with a single dose of 250 mg/kg 4M2-C12-hIgG1 ([1] of Example 2.2) or an equal volume of PBS. Blood samples were obtained at 6, 24, 96 and 168 hours, and analysed for numbers of different types of white blood cells using HM5 Hematology Analyser. Blood samples were also analysed for correlates hepatotoxicity, nephrotoxicity and pancreas toxicity.
[1767] Representative results are shown in the tables of
[1768] Administration of 4M2-012-hIgG1 was not found to be associated with significant toxicity, and did not significantly alter numbers of cell types in blood.