# US 2021/0277112 A1 · Antibodies Against ILT2 and Use Thereof

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- Number: US 2021/0277112 A1
- Document ID: US20210277112A1
- Status: Published application
- Kind: A1
- Application number: 17/224,016
- Filed: 2021-04-06
- Published: 2021-09-09
- Earliest claimed: 2019-08-12
- Assignees: Biond Biologics Ltd.
- Applicants: Biond Biologics Ltd.
- Inventors: Ilana MANDEL; Tsuri PERETZ; Dana HAVES ZIV; Ilana GOLDSHTEIN; Dror ALISHEKEVITZ; Anna FRIDMAN-DROR; Motti HAKIM; Avidor SHULMAN; Yair SAPIR; Tehila BEN-MOSHE
- Web page: https://patentgrep.com/patent/US20210277112A1

## CPC

- C07K 16/2803: {against the immunoglobulin superfamily}
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61P 35/00: Antineoplastic agents
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- C07K 16/2887: {against CD20}
- A61K 2039/505: {comprising antibodies}
- A61K 2039/507: {Comprising a combination of two or more separate antibodies}
- C07K 2317/34: Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
- C07K 2317/52: Constant or Fc region; Isotype
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/71: Decreased effector function due to an Fc-modification
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2317/732: Antibody-dependent cellular cytotoxicity [ADCC]
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

The present invention provides monoclonal antibodies, or antigen-binding portions thereof, against ILT2, as well as pharmaceutical compositions comprising same and methods of producing same. Also provided are methods of treating cancer comprising administering the antibodies or compositions of the invention. Methods of treating cancer, combination treatments, and patient selection are also provided.

## Claims

### Claim 1

A monoclonal antibody or antigen-binding fragment comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein:
a. CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 13 (SGYYWN), CDR-H2 comprises the amino acid sequence as set forth in SEQ ID NO: 14 (YISYDGSNNYNPSLKN), CDR-H3 comprises the amino acid sequence as set forth in SEQ ID NO: 15 (GYSYYYAMDX), CDR-L1 comprises the amino acid sequence as set forth in SEQ ID NO: 16 (RTSQDISNYLN), CDR-L2 comprises the amino acid sequence as set forth in SEQ ID NO: 17 (YTSRLHS), and CDR-L3 comprises the amino acid sequence as set forth in SEQ ID NO: 18 (QQGNTLPT), wherein said X is selected from A, C and S;
b. CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 1 (DHTIH), CDR-H2 comprises the amino acid sequence as set forth in SEQ ID NO: 2 (YIYPRDGSTKYNEKFKG), CDR-H3 comprises the amino acid sequence as set forth in SEQ ID NO: 3 (TWDYFDY), CDR-L1 comprises the amino acid sequence as set forth in SEQ ID NO: 4 (RASESVDSYGNSFMH), CDR-L2 comprises the amino acid sequence as set forth in SEQ ID NO: 5 (RASNLES), and CDR-L3 comprises the amino acid sequence as set forth in SEQ ID NO: 6 (QQSNEDPYT); or
c. CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 7 (GYTFTSYGIS), CDR-H2 comprises the amino acid sequence as set forth in SEQ ID NO: 8 (EIYPGSGNSYYNEKFKG), CDR-H3 comprises the amino acid sequence as set forth in SEQ ID NO: 9 (SNDGYPDY), CDR-L1 comprises the amino acid sequence as set forth in SEQ ID NO: 10 (KASDHINNWLA), CDR-L2 comprises the amino acid sequence as set forth in SEQ ID NO: 11 (GATSLET), and CDR-L3 comprises the amino acid sequence as set forth in SEQ ID NO: 12 (QQYWSTPWT).

### Claim 2 (depends on 1)

The antibody or antigen-binding fragment of claim 1, comprising a heavy chain comprising an amino acid sequence selected from:
SEQ ID NO: 19
(QVQLQQSDAELVKPGASVKISCKVSGYTFTDHTIHWMKQRPEQGLEWI
GYIYPRDGSTKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCA
RTWDYFDYWGQGTTLTVSS),
SEQ ID NO: 21
(QVQLQQSGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWV
GETYPGSGNSYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCA
RSNDGYPDYWGQGTTLTVSS)
and
SEQ ID NO: 23
(DVQLQGSGPGLVKPSETLSLTCSVTGYSITSGYYWNWIRQFPGKKLEW
MGYISYDGSNNYNPSLKNRITISRDTSKNQFSLKLNSVTAADTATYYCA
HGYSYYYAMDXWGQGTSVTVSS)[[]],
wherein said X is selected from A, C and S.

### Claim 3 (depends on 1)

The antibody or antigen-binding fragment of claim 1, comprising a light chain comprising an amino acid sequence selected from:
SEQ ID NO: 20
(DIVLTQSPASLAVSLGQRATISCRASESVDSYGNSFMHWYQQKPGQPP
KLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNE
DPYTFGGGTKLEIK),
SEQ ID NO: 22
(DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLI
SGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPWT
FGGGTKLEIK),
SEQ ID NO: 24
(DIQMTQSPSSLSASVGDRVTITCRTSQDISNYLNWYQQKPGKAVKLLI
SYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPTF
GQGTKLEIK)
and
SEQ ID NO: 45
(DIQMTQTTSSLSASLGDRVTISCRTSQDISNYLNWYQQKPDGTVKLLI
SYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPTF
GSGTKLEIK).

### Claim 4 (depends on 1)

The antibody or antigen-binding fragment of claim 1, wherein said antibody or antigen-binding fragment is humanized and said X is selected from A and S.

### Claim 5 (depends on 4)

The antibody or antigen-binding fragment of claim 4, wherein said X is A, SEQ ID NO: 15 is GYSYYYAMDA (SEQ ID NO: 25) and SEQ ID NO: 23 is
(SEQ ID NO: 28)
DVQLQGSGPGLVKPSETLSLTCSVTGYSITSGY)
(WNWIRQFPGKKLEWMGYISYDGSN
NYNPSLKNRITISRDTSKNQFSLKLNSVTAADTAT
YYCAHGYSYYYAMDAWGQGTSVTVSS.

### Claim 6

A monoclonal antibody or antigen-binding fragment
that binds a human leukocyte immunoglobulin-like receptor subfamily B member 1 (ILT2) epitope within a sequence of human ILT2 selected from VKKGQFPIPSITWEH (SEQ ID NO: 41), LELVVTGAYIKPTLS (SEQ ID NO: 42), VILQCDSQVAFDGFS (SEQ ID NO: 43) and WYRCYAYDSNSPYEW (SEQ ID NO: 44), or
that binds ILT2 and inhibits direct interaction between said ILT2 and beta-2-microglobulin (B2M).

### Claim 7 (depends on 6)

The antibody or antigen-binding fragment of claim 6, wherein said epitope is a 3-dimensional epitope comprising SEQ ID NO: 41, 42, 43 and 44.

### Claim 8

(canceled)

### Claim 9 (depends on 6)

The antibody or antigen-binding fragment of claim 6, wherein said antibody or antigen-binding fragment inhibits interaction of said ILT2 and an HLA protein or MHC-I protein via said inhibition of ILT2 direct interaction with B2M.

### Claim 10 (depends on 9)

The antibody or antigen-binding fragment of claim 9, wherein said HLA is HLA-G.

### Claim 11 (depends on 6)

The monoclonal antibody or antigen-binding fragment of claim 6, wherein the antibody or antigen-binding fragment induces in a subject suffering from cancer at least three of:
a. increased natural killer (NK) cell cytotoxicity;
b. increased T cell cytotoxicity, proliferation, or both;
c. increased macrophage phagocytosis, increased generation of M1 inflammatory macrophages, decreased generation of M2 suppressor macrophages or a combination thereof; and
d. increased dendritic cell homing to a tumor of said cancer, increased dendritic cell activation or a combination thereof.

### Claim 12 (depends on 11)

The antibody or antigen-binding fragment of claim 11, wherein said cancer is a HLA-G or MHC-I expressing cancer.

### Claim 13 (depends on 1)

A method of inducing/enhancing an anti-tumor T-cell response, increasing T-cell proliferation, reducing cancer-induced suppressor myeloid activity, increasing natural killer cell cytotoxicity, increasing macrophage phagocytosis, increasing generation of M1 inflammatory macrophages, decreasing generation of M2 suppressor macrophages, increasing dendritic cell number in a tumor microenvironment, increasing dendritic cell activation, treating an HLA-G expressing cancer, or treating a MHC-I expressing cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the antibody or antigen-binding fragment of claim 1.

### Claim at position 14

14-15. (canceled)

### Claim 16 (depends on 1)

A method of treating an HLA-G or MHC-I expressing cancer in a subject in need thereof, the method comprising administering to said subject a pharmaceutical composition comprising an antibody or antigen-binding fragment of claim 1.

### Claim 17 (depends on 16)

The method of claim 16, further comprising administering to said subject an opsonizing agent.

### Claim 18 (depends on 17)

The method of claim 17, wherein said opsonizing agent is an EGFR inhibitor, optionally wherein said EGFR inhibitor is cetuximab.

### Claim 19 (depends on 17)

The method of claim 17, further comprising administering to said subject an anti-PD-L1/PD-1 based immunotherapy.

### Claim 20 (depends on 1)

A method of treating an HLA-G or MHC-I expressing cancer in a subject in need thereof, the method comprising:
a. confirming expression of ILT2 or soluble HLA-G in said subject is above a predetermined threshold; and
b. administering to said subject the antibody or antigen-binding fragment of claim 1; thereby treating a cancer in a subject.

### Claim 21 (depends on 20)

The method of claim 20, wherein said confirming comprises measuring expression of said ILT2 or soluble HLA-G in said subject before said administering.

### Claim 22 (depends on 20)

The method of claim 20, comprising confirming expression of ILT2 and wherein said expression of ILT2 is in an immune cell of said subject.

### Claim 23 (depends on 22)

The method of claim 22, wherein said immune cell is selected from a peripheral blood immune cell and an intratumor immune cell.

### Claim 24 (depends on 22)

The method of claim 22, wherein said immune cell is selected from a CD8 positive T cell, a macrophage, an NK cell and a TEMRA cell.

### Claim 25 (depends on 24)

The method of claim 24, wherein said immune cell is a peripheral blood CD8 positive T cell.

### Claim 26 (depends on 20)

The method of claim 20, comprising confirming expression of soluble HLA-G.

### Claim at position 26

27-28. (canceled)

### Claim 29 (depends on 1)

A method of increasing efficacy of an anti-PD-L1/PD-1 based therapy against a cancer cell expressing HLA-G, MHC-I or both, the method comprising contacting said cancer cell with the antibody or antigen-binding fragment of claim 1.

### Claim at position 28

30-32. (canceled)

### Claim 33 (depends on 27)

The method of claim 27, wherein said anti-PD-L1/PD-1 based immunotherapy is an anti-PD-1 blocking antibody.

### Claim at position 30

34-37. (canceled)

### Claim 38 (depends on 1)

A pharmaceutical composition comprising an antibody or antigen-binding fragment of claim 1.

### Claim 39

A method for producing an agent, the method comprising:
A. a) obtaining an agent that binds to an ILT2 extracellular domain or fragment thereof,
testing an ability of said agent to induce at least two of: increased phagocytosis of a cancer cell by macrophages, increased T cell activity against a cancer cell, increased generation of M1 macrophages, reduced generation of M2 macrophages, increased recruitment of dendritic cells to a tumor microenvironment, increased dendritic cell activation and increased natural killer (NK) cell cytotoxicity against a cancer cell and
selecting at least one agent that induces at least two of said increased phagocytosis, said increased activity, said increased generation, said reduced generation, said recruitment, said increased activation, said decreased activity and said increased cytotoxicity; or
b) culturing a host cell comprising one or more vectors comprising a nucleic acid sequence encoding an agent, wherein the nucleic acid sequence is that of an agent that was selected by:
i. obtaining an agent that binds to an ILT2 extracellular domain or fragment thereof;
ii. testing an ability of said agent to induce at least two of: increased phagocytosis of a cancer cell by macrophages, increased T cell activity against a cancer cell, increased generation of M1 macrophages, reduced generation of M2 macrophages, increased recruitment of dendritic cells to a tumor microenvironment, increased dendritic cell activation and increased natural killer (NK) cell cytotoxicity against a cancer cell; and
iii. selecting at least one agent that increases at least two of said increased phagocytosis, said increased activity, said increased generation, said reduced generation, said recruitment, said increased activation, said decreased activity and said increased cytotoxicity; thereby producing an agent, or
B. a) obtaining an agent that binds to an ILT2 extracellular domain or fragment thereof,
testing an ability of said agent to increase efficacy of an anti-PD-L1/PD-1 based therapy against a cancer cell and selecting at least one agent that increases the efficacy of an anti-PD-L1/PD-1 based therapy; or
b) culturing a host cell comprising one or more vectors comprising a nucleic acid sequence encoding an agent, wherein the nucleic acid sequence is that of an agent that was selected by:
i. obtaining an agent that binds to an ILT2 extracellular domain or fragment thereof;
ii. testing an ability of said agent to increase efficacy of an anti-PD-L1/PD-1 based therapy against a cancer cell; and
iii. selecting at least one agent that increases efficacy of an anti-PD-L1/PD-1 based therapy against a cancer cell;
thereby producing an agent.

### Claim at position 33

40-47. (canceled)

### Claim 48

A method for producing an agent, the method comprising:
A. obtaining an agent that binds to an ILT2 extracellular domain or fragment thereof,
testing an ability of said agent to inhibit interaction between ILT2 and B2M and
selecting at least one agent that inhibits interaction between ILT2 and B2M; or
b) culturing a host cell comprising one or more vectors comprising a nucleic acid sequence encoding an agent, wherein the nucleic acid sequence is that of an agent that was selected by:
i. obtaining an agent that binds to an ILT2 extracellular domain or fragment thereof;
ii. testing an ability of said agent to inhibit interaction between ILT2 and B2M; and
iii. selecting at least one agent that inhibits interaction between ILT2 and B2M;
thereby producing an agent, or
B. a) obtaining an agent that binds to an ILT2 epitope within a sequence of human ILT2 selected from SEQ ID NO: 41, 42, 43 and 44, or
b) culturing a host cell comprising one or more vectors comprising a nucleic acid sequence encoding an agent, wherein the nucleic acid sequence is that of an agent that was selected by obtaining an agent that binds to an ILT2 epitope within a sequence of human ILT2 selected from SEQ ID NO: 41, 42, 43 and 44; thereby producing an agent.

### Claim 49

(canceled)

### Claim 50 (depends on 1)

A nucleic acid molecule encoding an antibody or antigen-binding fragment of claim 1.

### Claim 51 (depends on 50)

The nucleic acid molecule of claim 50, wherein said nucleic acid molecule is an expression vector.