Antibodies specific for immunoglobulin-like transcript 3 (ILT3) and uses thereof

Abstract

Humanized, non-hybrid monoclonal antibodies are described that are specific for immunoglobulin-like transcript 3 (ILT3), also referred to as leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4).

Description

[0001] This application is a divisional application of the patent application with the application date of November 15, 2018, application number 201880086998.8, and title "Antibodies Specific for Immunoglobulin-like Transcript 3 (ILT3) and Their Use". Background of the Invention (1) Field of the Invention

[0003] The present invention provides non-confounding monoclonal antibodies specific for the inhibitory receptor immunoglobulin-like transcript 3 (ILT3) expressed on the surface of myeloid immune cells.

[0004] (2) Description of Related Art

[0005] Immunoglobulin-like transcript 3 (ILT3), designated CD85k, also known as leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4) and leukocyte immunoglobulin-like receptor 5 (LIR-5), is a type I membrane protein that contains a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) motif and is involved in the downregulation of the immune response (Cella et al., J Exp Med. 185(10):1743-51 (1997); Samarisdis et al., Eur J Immunol 27(3):660-665 (1997)). The expression of ILT3 is upregulated on tolerogenic dendritic cells. The gene is a member of the leukocyte immunoglobulin-like receptor (LIR) family and is found in a gene cluster at chromosomal region 19q13.4. The encoded protein belongs to subfamily B of the LIR receptors and contains two or four extracellular immunoglobulin domains, a transmembrane domain, and two to four ITIMs.

[0006] ILT3 is selectively expressed by myeloid antigen-presenting cells (APCs), such as monocytes, macrophages, and dendritic cells, such as monocyte-derived dendritic cells differentiated in the presence of IL-10 or vitamin D3. ILT3 consists of 447 amino acids and has a predicted molecular weight of approximately 47 kD. The amino-terminal portion of ILT3 begins with a 23-amino acid hydrophobic signal peptide, followed by an extracellular domain containing two C2-type immunoglobulin superfamily domains, having the amino acid sequence shown in SEQ ID NO:1, minus the C-terminal His tag. (The extracellular domain of rhesus ILT3 has the amino acid sequence shown in SEQ ID NO:2). The putative transmembrane domain of ILT3 consists of 21 amino acids, followed by a long cytoplasmic region of 167 amino acids, which is characterized by the presence of motifs spaced 26 amino acid residues apart, reminiscent of the ITIM motif (Natural-Killer cell Ig Receptor) identified in KIR as the binding site for the protein tyrosine phosphatase SHP-1. ILT3 is expressed on immune cells, where it binds to MHC class I molecules on antigen-presenting cells and transduces a negative signal that inhibits the stimulation of the immune response. The receptor may also play a role in antigen capture and presentation. ILT3 is thought to control the inflammatory response and cytotoxicity to help focus the immune response and limit autoreactivity. Multiple transcript variants encoding different isoforms of ILT3 have been identified.

[0007] Patent publications that disclose the use of antibodies to modulate ILT3 activity and for applications in inhibiting transplant rejection or for the treatment of cancer or infectious diseases include US Publication Nos. 20090202544, 20150110714, 20150139986, and 20170267759; and International Publication Nos. WO2013043569, WO2013181438, WO2014116846, WO2016049641, WO2016127427, WO2018089300, and WO2018148494. Of interest is International Publication No. WO2017015227, which discloses CD166, also known as Activated Leukocyte Cell Adhesion Molecule (ALCAM), which is a ligand for ILT3 and, in some embodiments, provides methods for treating cancer that include antibodies against CD166 or ALCAM. Also of interest are US Patent Nos. 7777008 and 8901281, which disclose the monoclonal antibody 9B11 for use in a variety of treatments, where it is desired to upregulate the immune system for anti-cancer treatment and downregulate the immune system for inhibiting transplant rejection.

[0008] Although patents disclose anti-ILT3 antibodies, in some cases no specific antibody is disclosed or multiple specific antibodies are disclosed, which in some cases are shown to be promiscuous and cross-react with one or more ILT3-related receptors such as LILRA6 and ILT8. Promiscuous anti-ILT3 antibodies can have off-target effects, which can have undesirable effects that make their use for therapeutic applications contraindicated. Thus, there is a need for antibodies and antigen-binding fragments that specifically bind ILT3 and have no measurable promiscuity with other related receptors. SUMMARY OF THE INVENTION

[0009] The present invention provides monoclonal antibodies and antigen-binding fragments that specifically bind immunoglobulin-like transcript 3 (ILT3) and have no measurable binding to closely related proteins (e.g., ILT5, ILT7, ILT8, or ILT11), as determined by: (i) cell ELISA using 10 μg / mL of the antibody or antigen-binding fragment, or (ii) Biacore using 10 μg / mL of the antibody or antigen-binding fragment. In certain embodiments, the antibodies and antigen-binding fragments specifically bind both human ILT3 and cynomolgus ILT3. These antibodies and antigen-binding fragments are capable of antagonizing ILT3 activity, thereby enhancing the activation of dendritic cells and the priming of T cells. Tolerized dendritic cells and myeloid-derived suppressor cells (MDSC) also respond to these antibodies. In addition, these antibodies in a humanized NSG TM mouse model system (Jackson Laboratory, Bar Harbor, Maine) in vivo studies show that these antibodies can have the ability to reduce tumor burden and shift the cell phenotype to a more activated state.

[0010] In clinical trial samples, ILT3 expression, such as PD-L1, LAG-3, and GEP signatures, was found to be associated with responsiveness to anti-PD-1 antibodies (pembrolizumab). In certain cancer types, soluble ILT3 in the circulation is also increased. In summary, the anti-ILT3 antibodies of the present invention can be used to treat specific cancers, either as a single therapy or in combination with anti-PD-1 and / or anti-PD-L1 antibodies to enhance responsiveness to anti-PD-1 or anti-PD-L1 antibodies, particularly in the treatment of cancers that are unresponsive to anti-PD-1 or anti-PD-L1 monotherapy. In certain embodiments, the present invention provides chimeric or humanized anti-ILT3 antibodies. In certain embodiments, the antibody can be a fully human antibody that competes with the antibodies disclosed herein for binding to the ILT3 epitopes disclosed herein.

[0011] The present invention provides an antibody or antigen-binding fragment comprising a heavy chain variable V having heavy chain complementarity determining regions (HC-CDR) 1, 2, and 3 HOne, two, or three complementarity determining regions (CDRs) of a domain, and a light chain variable domain V having LC-CDR1, 2, and 3 L One, two, or three CDRs thereof, wherein the antibody or antigen-binding fragment is capable of specifically binding to human ILT3, and the binding of the antibody or antigen-binding fragment can be determined by cell ELISA or Biacore assay.

[0012] In another embodiment, the antibody or antigen-binding fragment binds to an epitope on human ILT3 or competes with the disclosed antibody for binding to an epitope on human ILT3, wherein the epitope comprises at least one amino acid within one or more of the amino acid sequences set forth in SEQ ID NOs: 3, 4, 5, 6, 7, and 8. In another embodiment, the antibody or antigen-binding fragment binds to an epitope on human ILT3 or competes with the disclosed antibody for binding to an epitope on human ILT3, wherein the epitope comprises one or more of the amino acid sequences set forth in SEQ ID NOs: 3, 4, 5, 6, 7, and 8. In another embodiment, the antibody or antigen-binding fragment binds to an epitope on human ILT3 or competes with the disclosed antibody for binding to an epitope on human ILT3, wherein the epitope comprises the amino acid sequences set forth in SEQ ID NOs: 3, 4, 5, 6, 7, and 8. In a particular embodiment, the epitope is determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis.

[0013] The present invention further provides an antibody or antigen-binding fragment that binds to human ILT3, which comprises a heavy chain (HC), wherein the heavy chain variable domain (V H ) comprises a heavy chain complementarity determining region (HC-CDR) 3 having an amino acid sequence selected from SEQ ID NOs: 22, 49, 57, 65, 73, 81, 89, 97, and 105, or an amino acid sequence having 3, 2, or 1 difference from the amino acid sequences selected from SEQ ID NOs: 22, 49, 57, 65, 73, 81, 89, 97, and 105. In some embodiments, the amino acid sequence difference is a conservative change / substitution. In a particular embodiment, the antibody or antigen-binding fragment that binds to human ILT3 comprises a heavy chain (HC), wherein the heavy chain variable domain (V H ) comprises a heavy chain complementarity determining region (HC-CDR) 3 having an amino acid sequence selected from SEQ ID NOs: 23, 49, 57, 65, 73, 81, 89, 97, and 105, or an amino acid sequence having 3, 2, or 1 difference from the amino acid sequences selected from SEQ ID NOs: 23, 49, 57, 65, 73, 81, 89, 97, and 105. In a particular embodiment, the amino acid sequence difference is a conservative change / substitution.

[0014] In another embodiment, the antibody or antigen-binding fragment binds to an epitope on human ILT3 or competes with the disclosed antibodies for binding to an epitope on human ILT3, wherein the epitope comprises at least one amino acid from one or more of the amino acid sequences set forth in SEQ ID NOs: 3, 4, 5, 6, 7, and 8. In another embodiment, the antibody or antigen-binding fragment binds to an epitope on human ILT3 or competes with the disclosed antibodies for binding to an epitope on human ILT3, wherein the epitope comprises one or more of the amino acid sequences set forth in SEQ ID NOs: 3, 4, 5, 6, 7, and 8. In another embodiment, the antibody or antigen-binding fragment binds to an epitope on human ILT3 or competes with the disclosed antibodies for binding to an epitope on human ILT3, wherein the epitope comprises the amino acid sequences set forth in SEQ ID NOs: 3, 4, 5, 6, 7, and 8. In a specific embodiment, the epitope is determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis.

[0015] The present invention further provides an antibody or antigen-binding fragment that binds to human ILT3, comprising (a) a heavy chain (HC) having a variable domain (V H ), which comprises a variable domain complementarity-determining region (HC-CDR) 1 having the amino acid sequence set forth in SEQ ID NO: 17, 47, 55, 63, 71, 79, 87, 95, or 103; an HC-CDR2 having the amino acid sequence set forth in SEQ ID NO: 18, 48, 56, 64, 72, 80, 88, 96, or 104; an HC-CDR3 having the amino acid sequence set forth in SEQ ID NO: 23, 49, 57, 65, 73, 81, 89, 97, or 105; and variants thereof wherein one or more of the HC-CDRs have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof; and (b) a light chain (LC) having a variable domain (V L ), which comprises a variable domain complementarity-determining region (LC-CDR) 1 having the amino acid sequence set forth in SEQ ID NO: 27, 50, 58, 66, 74, 82, 90, 98, or 106; an LC-CDR2 having the amino acid sequence set forth in SEQ ID NO: 43, 51, 59, 67, 75, 83, 91, 99, or 107; an LC-CDR3 having the amino acid sequence set forth in SEQ ID NO: 44, 60, 68, 76, 84, 92, 100, or 108; and variants thereof wherein one or more of the LC-CDRs have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof. In a specific embodiment, the amino acid sequence differences are conservative changes / substitutions.

[0016] In another embodiment of the antibody or antigen-binding fragment, HC-CDR1 has the amino acid sequence shown in SEQ ID NO:17; HC-CDR2 has the amino acid sequence shown in SEQ ID NO:19, 20 or 21; HC-CDR3 has the amino acid sequence shown in SEQ ID NO:23; and LC-CDR1 has the amino acid sequence shown in SEQ ID NO:34, 35, 36, 37, 38, 39, 40, 41 or 42; LC-CDR2 has the amino acid sequence shown in SEQ ID NO:43; and LC-CDR3 has the amino acid sequence shown in SEQ ID NO:44; and variants thereof in which one or more of the HC-CDRs and LC-CDRs have one, two or three amino acid substitutions, additions, deletions or combinations thereof. In certain embodiments, the amino acid sequence differences are conservative changes / substitutions.

[0017] In another embodiment of the antibody or antigen-binding fragment, HC-CDR1 has the amino acid sequence shown in SEQ ID NO:17; HC-CDR2 has the amino acid sequence shown in SEQ ID NO:20; HC-CDR3 has the amino acid sequence shown in SEQ ID NO:23; and LC-CDR1 has the amino acid sequence shown in SEQ ID NO:41; LC-CDR2 has the amino acid sequence shown in SEQ ID NO:43; and LC-CDR3 has the amino acid sequence shown in SEQ ID NO:44; and variants thereof in which one or more of the HC-CDRs and LC-CDRs have one, two or three amino acid substitutions, additions, deletions or combinations thereof. In certain embodiments, the amino acid sequence differences are conservative changes / substitutions.

[0018] In another embodiment of the antibody or antigen-binding fragment, the antibody or antigen-binding fragment comprises (a) V H , which has a framework selected from the human V H 1, V H 2, V H 3, V H 4, V H 5, and V H 6 families, and variants thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof; and (b) V L , which has a framework selected from the human V κ 1, V κ 2, V κ 3, V κ 4, V κ 5, V κ 6, V λ 1, V λ 2, Vλ 3. V λ 4. V λ 5. V λ 6. V λ 7. V λ 8. V λ 9. and V λ 10 families, and frameworks of variants thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof. In certain embodiments, the amino acid sequence differences are conservative alterations / substitutions.

[0019] In certain embodiments, the antibody or antigen-binding fragment comprises (a) V H , which has a human V H 1 family framework or a variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof; and (b) V L , which has a human V κ 5 family framework or a variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof. In certain embodiments, the amino acid sequence differences are conservative alterations / substitutions.

[0020] In another embodiment of the antibody, the antibody comprises a human IgG1, IgG2, IgG3, or IgG4 HC constant region or a variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native IgG1, IgG2, IgG3, or IgG4 isotype HC constant domain. In certain aspects, the constant domain may comprise a C-terminal lysine or may lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide.

[0021] In certain embodiments, the heavy chain constant domain has the human IgG1 isotype, which has been modified to have reduced or minimal effector function. In a further aspect, minimal effector function is due to an effector-less Fc mutation, which may comprise or consist of mutations N297A or D265A / N297A as determined using Kabat numbering, in which case minimal effector function is due to aglycosylation (see, e.g., the amino acid sequence shown in SEQ ID NO: 211, where the N297A mutation corresponds to amino acid position 180; the D265A mutation (if present) would correspond to amino acid position 148). In certain aspects, IgG1 has been modified to comprise or consist of mutations L234A, L235A, and D265S as determined using Kabat numbering to render the Fc effector-less (see, e.g., the amino acid sequences shown in SEQ ID NO: 12 or 13, where the L234A, L235A, and D265S mutations correspond to amino acid positions 117, 118, and 148, respectively).

[0022] In another aspect, the HC constant domain has the human IgG4 isotype, and the isotype further comprises a proline substitution for the serine residue at position 228 (EU numbering), which corresponds to position 108 (serine at position 108) of SEQ ID NO: 9 or 10.

[0023] In another embodiment of the antibody or antigen-binding fragment, the antibody comprises a human κ or λ LC constant domain or a variant thereof that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native human κ or λ LC constant domain. In certain embodiments, the amino acid sequence differences are conservative changes / substitutions.

[0024] In another embodiment of the antibody or antigen-binding fragment, the antibody comprises (i) a V H that has a framework selected from the human V H 1, V H 2, V H 3, V H 4, V H 5, and V H 6 families and a variant of the human IgG1 or IgG4 HC constant domain that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native human IgG1 or IgG4 isotype HC constant domain; and (ii) a V L that has a framework selected from the human V κ 1, V κ 2, Vκ 3. V κ 4. V κ 5. V κ 6. V λ 1. V λ 2. V λ 3. V λ 4. V λ 5. V λ 6. V λ 7. V λ 8. V λ 9 and V λ The framework of the 10 family and the human κ or λ LC constant domain or a variant thereof that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native human κ or λ LC constant domain. In certain embodiments, the amino acid sequence differences are conservative changes / substitutions.

[0025] In another embodiment of the antibody or antigen-binding fragment, the antibody comprises (i) a V having a human V H framework of the 2 family H and a V having a human V κ framework of the 5 family L ; (ii) a human IgG1 or IgG4 HC constant domain or a variant thereof that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native human IgG1 or IgG4 isotype HC constant domain; and (iii) a human κ LC constant domain or a variant thereof that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native human κ LC constant domain. In certain embodiments, the amino acid sequence differences are conservative changes / substitutions.

[0026] In another embodiment of the antibody or antigen-binding fragment, the antibody comprises (i) a V having a human V H framework of the 1 family H and a human V having a human V κ framework of the 5 family L ; (ii) a human IgG4 HC constant domain or a variant thereof that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native human IgG4 isotype HC constant domain; and (iii) a human κ LC constant domain or a variant thereof that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native human κ LC constant domain. In certain embodiments, the amino acid sequence differences are conservative changes / substitutions.

[0027] In another embodiment of the antibody or antigen-binding fragment, the antibody or antigen-binding fragment comprises V having the amino acid sequences set forth in SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 45 and SEQ ID NO: 46; SEQ ID NO: 53 and SEQ ID NO: 54; SEQ ID NO: 61 and SEQ ID NO: 62; SEQ ID NO: 69 and SEQ ID NO: 70; SEQ ID NO: 77 and SEQ ID NO: 78; SEQ ID NO: 85 and SEQ ID NO: 86; SEQ ID NO: 93 and SEQ ID NO: 94; or SEQ ID NO: 101 and SEQ ID NO: 102, respectively H and V L .

[0028] In another embodiment of the antibody or antigen-binding fragment, the antibody or antigen-binding fragment comprises V having the amino acid sequence set forth in SEQ ID NO: 117, 118, 119, 123, 124, or 125 H ; and V having the amino acid sequence set forth in SEQ ID NO: 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, or 141 L .

[0029] In another embodiment of the antibody or antigen-binding fragment, the antibody or antigen-binding fragment comprises V having the amino acid sequence set forth in SEQ ID NO: 118 H ; and V having the amino acid sequence set forth in SEQ ID NO: 140 L .

[0030] In another embodiment of the antibody, the antibody comprises a HC constant domain comprising the amino acid sequence set forth in SEQ ID NO: 9, 10, 11, 12, or 13. In certain aspects, the HC constant domain comprising the amino acid sequence set forth in SEQ ID NO: 9, 11, 12, or 13 may lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide. In certain embodiments, the HC constant domain comprises the amino acid sequence set forth in SEQ ID NO: 10

[0031] In another embodiment of the antibody, the antibody comprises a LC constant domain comprising the amino acid sequence set forth in SEQ ID NO: 14

[0032] In another embodiment of the antibody, the antibody comprises a heavy chain (HC) that comprises the amino acid sequence of SEQ ID NO: 142, 143, 144, 148, 149, 150, 167, 168, 169, 170, 174, 175, 176, 177, 178, 182, 183, 184, 185, 186, 187, 191, 192, or 193. In certain aspects, the HC comprising the amino acid sequence set forth in SEQ ID NO: 142, 143, 144, 148, 149, 150, 167, 168, 169, 170, 174, or 175 may lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide. In certain embodiments, the HC comprises the amino acid sequence as set forth in SEQ ID NO: 143 or 177. In certain embodiments, the HC as set forth in SEQ ID NO: 177 further lacks a C-terminal glycine.

[0033] In another embodiment of the antibody, the antibody comprises a light chain (LC) that comprises the amino acid sequence set forth in SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, or 166. In certain embodiments, the LC comprises the amino acid sequence as set forth in SEQ ID NO: 165.

[0034] In another embodiment of the antibody, the antibody comprises an HC having the amino acid sequence as set forth in SEQ ID NO: 143 and an LC having the amino acid sequence as set forth in SEQ ID NO: 165. In certain aspects, the HC comprising the amino acid sequence as set forth in SEQ ID NO: 143 lacks a C-terminal lysine residue or a C-terminal glycine-lysine dipeptide.

[0035] The present invention further provides a chimeric, humanized or recombinant human antibody or antigen-binding fragment that binds to an epitope on human ILT3, wherein the epitope comprises at least one amino acid within the amino acid sequences set forth in SEQ ID NO: 3, 4, 5, 6, 7, and 8. In another embodiment, the chimeric, humanized or recombinant human antibody or antigen-binding fragment binds to an epitope on human ILT3, the epitope comprising the amino acid sequences set forth in SEQ ID NO: 3, 4, 5, 6, 7, and 8. In these embodiments, the epitope is determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis.

[0036] The present invention further provides a chimeric, humanized or recombinant human antibody or antigen-binding fragment that binds to ILT3, wherein the binding cross-blocks the antibody or competes with the antibody for binding, and the antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 15 and a light chain having the amino acid sequence shown in SEQ ID NO: 16. In another embodiment, the cross-blocking antibody or the chimeric, humanized or recombinant human antibody or antigen-binding fragment that competes with the antibody binds to an epitope on ILT3 comprising the amino acid sequences shown in SEQ ID NO: 3, 4, 5, 6, 7 and 8, and the antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 15 and a light chain having the amino acid sequence shown in SEQ ID NO: 16.

[0037] The present invention further provides a composition comprising one or more of any of the antibodies or antigen-binding fragments disclosed or claimed herein and a pharmaceutically acceptable carrier.

[0038] The present invention further provides a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment disclosed or claimed herein sufficient to treat the cancer in the subject.

[0039] In another embodiment, the cancer is pancreatic cancer, melanoma, breast cancer, lung cancer, head and neck cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma or blood tissue cancer.

[0040] The present invention also provides a method for treating cancer in a subject, which comprises administering to the subject a combination of an antibody or antigen-binding fragment disclosed herein and one or more inhibitors or antagonists of PD-1, PD-L1 and / or PD-L2 simultaneously or sequentially. In one embodiment, the antagonist of PD-1 is an antibody or an antigen-binding fragment thereof that binds to human PD-1 and blocks the binding of PD1 to human PD-L1 and PD-L2. In one embodiment, the antagonist of PD-L1 or PD-L2 is an antibody or an antigen-binding fragment thereof that binds to human PD-L1 or PD-L2 and blocks the binding of human PD-L1 or PD-L2 to PD1.

[0041] In another embodiment, the anti-PD1 antagonist is an anti-PD-1 antibody, which is nivolumab, pembrolizumab, cemiplimab, or pidilizumab, and the PD-L1 inhibitor is durvalumab, atezolizumab, avelumab, YW243.55.S70, MPDL3280A, MEDI-4736, MSB-0010718C, or MDX-1105.

[0042] The present invention further provides an antibody or antigen-binding fragment disclosed or claimed herein for treating cancer in a subject.

[0043] In another embodiment, the cancer is pancreatic cancer, melanoma, breast cancer, lung cancer, head and neck cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain cancer or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma or hematological tissue cancer.

[0044] The present invention further provides an antibody or antigen-binding fragment disclosed or claimed herein for treating cancer in a subject, wherein the treatment further comprises one or more inhibitors or antagonists of PD-1, PD-L1 and / or PD-L2.

[0045] In one embodiment, the antagonist of PD-1 is an antibody or an antigen-binding fragment thereof that binds to human PD-1 and blocks the binding of PD1 to PD-L1 and PD-L2.

[0046] In one embodiment, the antagonist of PD-L1 or PD-L2 is an antibody or an antigen-binding fragment thereof that binds to human PD-L1 or PD-L2 and blocks the binding of human PD-L1 or PD-L2 to PD1.

[0047] In another embodiment, the anti-PD-1 antibody is nivolumab, pembrolizumab, cemiplimab, or pidilizumab, and the PD-L1 inhibitor is durvalumab, atezolizumab, avelumab, YW243.55.S70, MPDL3280A, MEDI-4736, MSB-0010718C, or MDX-1105.

[0048] The present invention further provides the use of an antibody or antigen-binding fragment disclosed or claimed herein for treating cancer.

[0049] The present invention further provides the use of an antibody or antigen-binding fragment disclosed or claimed herein in the preparation of a medicament for treating cancer.

[0050] In another embodiment, the cancer is pancreatic cancer, melanoma, breast cancer, lung cancer, head and neck cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain cancer or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma or hematological tissue cancer.

[0051] The present invention further provides a composition comprising any one of the above-mentioned antibodies or antigen-binding fragments and a pharmaceutically acceptable carrier. In a particular embodiment, the composition comprises a mixture of an antibody comprising a heavy chain with a C-terminal lysine and an antibody comprising a heavy chain lacking a C-terminal lysine. In a particular embodiment, the composition comprises an antibody disclosed herein, wherein the major antibody form comprises a heavy chain with a C-terminal lysine. In a particular embodiment, the composition comprises an antibody disclosed herein, wherein the major antibody form comprises a heavy chain lacking a C-terminal lysine. In a particular embodiment, the composition comprises an antibody disclosed herein, wherein approximately 100% of the antibodies in the composition comprise a heavy chain lacking a C-terminal lysine.

[0052] This application also includes the following embodiments.

[0053] 1. An antibody or antigen-binding fragment that binds to human immunoglobulin-like transcript 3 (ILT3), comprising:

[0054] a heavy chain (HC), wherein the heavy chain variable domain (V H ) comprises a heavy chain complementarity-determining region (HC-CDR) 3 having an amino acid sequence selected from SEQ ID NO: 22, 49, 57, 65, 73, 81, 89, 97, and 105, or having an amino acid sequence that differs from the amino acid sequence selected from SEQ ID NO: 22, 49, 57, 65, 73, 81, 89, 97, and 105 by 3, 2, or 1 amino acid.

[0055] 2. The antibody or antigen-binding fragment according to embodiment 1, wherein the antibody or antigen-binding fragment binds to an epitope on human ILT3, and wherein the epitope comprises at least one amino acid from one or more of the amino acid sequences set forth in SEQ ID NO: 3, 4, 5, 6, 7, and 8.

[0056] 3. An antibody or antigen-binding fragment that binds to human immunoglobulin-like transcript 3 (ILT3), comprising:

[0057] (a) A heavy chain (HC) having a variable domain (V H ), which comprises a variable domain complementarity determining region (HC-CDR) 1 having the amino acid sequence shown in SEQ ID NO: 17, 47, 55, 63, 71, 79, 87, 95 or 103; an HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 18, 48, 56, 64, 72, 80, 88, 96 or 104; an HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 23, 49, 57, 65, 73, 81, 89, 97 or 105; and variants thereof wherein one or more of said HC-CDRs have one, two or three amino acid substitutions, additions, deletions or combinations thereof; and

[0058] (b) A light chain (LC) having a variable domain (V L ), which comprises a variable domain complementarity determining region (LC-CDR) 1 having the amino acid sequence shown in SEQ ID NO: 27, 50, 58, 66, 74, 82, 90, 98 or 106; an LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 43, 51, 59, 67, 75, 83, 91, 99 or 107; an LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 44, 60, 68, 76, 84, 92, 100 or 108; and variants thereof wherein one or more of said LC-CDRs have one, two or three amino acid substitutions, additions, deletions or combinations thereof.

[0059] 4. The antibody or antigen-binding fragment according to embodiment 3, wherein

[0060] (a) said HC-CDR1 has the amino acid sequence shown in SEQ ID NO: 17; said HC-CDR2 has the amino acid sequence shown in SEQ ID NO: 19, 20 or 21; and said HC-CDR3 has the amino acid sequence shown in SEQ ID NO: 23; and

[0061] (b) said LC-CDR1 has the amino acid sequence shown in SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, 41 or 42; said LC-CDR2 has the amino acid sequence shown in SEQ ID NO: 43; and said LC-CDR3 has the amino acid sequence shown in SEQ ID NO: 44.

[0062] 5. The antibody or antigen-binding fragment according to embodiment 4, wherein

[0063] (a) The HC-CDR1 has the amino acid sequence shown in SEQ ID NO:17; the HC-CDR2 has the amino acid sequence shown in SEQ ID NO:20; and the HC-CDR3 has the amino acid sequence shown in SEQ ID NO:23; and

[0064] (b) The LC-CDR1 has the amino acid sequence shown in SEQ ID NO:41; the LC-CDR2 has the amino acid sequence shown in SEQ ID NO:43; and the LC-CDR3 has the amino acid sequence shown in SEQ ID NO:44.

[0065] 6. The antibody or antigen-binding fragment according to embodiment 3, 4 or 5, wherein the V H comprises a framework selected from human V H 1, V H 2, V H 3, V H 4, V H 5, and V H 6, and variants thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof; and, the V L comprises a framework selected from human V κ 1, V κ 2, V κ 3, V κ 4, V κ 5, V κ 6, V λ 1, V λ 2, V λ 3, V λ 4, V λ 5, V λ 6, V λ 7, V λ 8, V λ 9, and V λ 10, and variants thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof.

[0066] 7. The antibody or antigen-binding fragment according to embodiment 3, 4, 5 or 6, wherein the antibody comprises an HC having a human IgG1, IgG2, IgG3 or IgG4 HC constant region or a variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof compared to the amino acid sequence of the native IgG1, IgG2, IgG3 or IgG4 isotype constant domain.

[0067] 8. The antibody or antigen-binding fragment according to embodiment 6 or 7, wherein the antibody comprises an LC having a human κ or λ LC constant domain or a variant thereof that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native human κ or λ light chain constant domain.

[0068] 9. The antibody or antigen-binding fragment according to embodiment 5, wherein the antibody comprises:

[0069] (i) V H having a framework selected from human V H 1, V H 2, V H 3, V H 4, V H 5, and V H 6 and a human IgG1 or IgG4 HC constant domain or a variant thereof that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native IgG1 or IgG4 isotype HC constant domain; and,

[0070] (ii) V L having a framework selected from human V κ 1, V κ 2, V κ 3, V κ 4, V κ 5, V κ 6, V λ 1, V λ 2, V λ 3, V λ 4, V λ 5, V λ 6, V λ 7, V λ 8, V λ 9, and V λ 10 and a human κ or λ LC constant domain or a variant thereof that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native human κ or λ LC constant domain.

[0071] 10. The antibody or antigen-binding fragment according to Embodiment 6, wherein the antibody or antigen-binding fragment comprises V having the amino acid sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 45 and SEQ ID NO: 46; SEQ ID NO: 53 and SEQ ID NO: 54; SEQ ID NO: 61 and SEQ ID NO: 62; SEQ ID NO: 69 and SEQ ID NO: 70; SEQ ID NO: 77 and SEQ ID NO: 78; SEQ ID NO: 85 and SEQ ID NO: 86; SEQ ID NO: 93 and SEQ ID NO: 94; or SEQ ID NO: 101 and SEQ ID NO: 102, respectively. H and V L .

[0072] 11. The antibody or antigen-binding fragment according to Embodiment 6, wherein the antibody or antigen-binding fragment comprises V having the amino acid sequence shown in SEQ ID NO: 117, 118, 119, 123, 124, or 125. H ; and V having the amino acid sequence shown in SEQ ID NO: 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, or 141. L .

[0073] 12. The antibody or antigen-binding fragment according to Embodiment 11, wherein the antibody or antigen-binding fragment comprises V having the amino acid sequence shown in SEQ ID NO: 118. H ; and V having the amino acid sequence shown in SEQ ID NO: 140. L .

[0074] 13. The antibody or antigen-binding fragment according to Embodiment 9, 10, 11, or 12, wherein the antibody comprises a heavy chain (HC) constant domain comprising the amino acid sequence shown in SEQ ID NO: 9, 10, 11, 12, or 13.

[0075] 14. The antibody or antigen-binding fragment according to Embodiment 9, 10, 11, or 12, wherein the antibody comprises a light chain (LC) constant domain comprising the amino acid sequence shown in SEQ ID NO: 14.

[0076] 15. The antibody or antigen-binding fragment according to embodiment 9, 10, 11, or 12, wherein the antibody comprises a heavy chain (HC), and the heavy chain comprises the amino acid sequence of SEQ ID NO: 142, 143, 144, 148, 149, 150, 167, 168, 169, 170, 174, 175, 176, 177, 178, 182, 183, 184, 185, 186, 187, 191, 192, or 193.

[0077] 16. The antibody or antigen-binding fragment according to embodiment 9, 10, 11, 12, 13, 14, or 15, wherein the antibody comprises a light chain (LC), and the light chain (LC) comprises the amino acid sequence shown in SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, or 166.

[0078] 17. The antibody or antigen-binding fragment according to embodiment 9, wherein the antibody comprises a heavy chain (HC) containing the amino acid sequence of SEQ ID NO: 143 and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 165, and variants thereof wherein the HC lacks the C-terminal lysine residue or C-terminal glycine-lysine.

[0079] 18. A chimeric, humanized, or recombinant human antibody or antigen-binding fragment that binds to an epitope on human immunoglobulin-like transcript 3 (ILT3), wherein the epitope comprises at least one amino acid from one or more of the amino acid sequences shown in SEQ ID NO: 3, 4, 5, 6, 7, and 8.

[0080] 19. A chimeric, humanized, or recombinant human antibody or antigen-binding fragment that cross-blocks the binding of an antibody to human immunoglobulin-like transcript 3 (ILT3) or competes with the antibody for binding to human immunoglobulin-like transcript 3 (ILT3), the antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 15 and a light chain containing the amino acid sequence of SEQ ID NO: 16.

[0081] 20. A composition comprising:

[0082] The antibody or antigen-binding fragment according to any one of embodiments 1-19 and a pharmaceutically acceptable carrier.

[0083] 21. A method for treating cancer in a subject, comprising administering to the subject the antibody or antigen-binding fragment according to any one of embodiments 1-19 or the composition according to embodiment 20.

[0084] 22. The method according to embodiment 21, wherein the cancer is pancreatic cancer, melanoma, breast cancer, lung cancer, head and neck cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma or blood tissue cancer.

[0085] 23. A method for treating cancer in a subject, comprising:

[0086] administering to the subject, simultaneously or sequentially, a combination of an antibody or antigen-binding fragment according to any one of embodiments 1-19 and one or more inhibitors or antagonists of PD-1, PD-L1, and / or PD-L2.

[0087] 24. The method according to embodiment 23, wherein the anti-PD-1 antibody is pembrolizumab, nivolumab, cemiplimab, and pidilizumab.

[0088] 25. The method according to embodiment 23, wherein the PD-L1 inhibitor is durvalumab, atezolizumab, avelumab, YW243.55.S70, MPDL3280A, MEDI-4736, MSB-0010718C, or MDX-1105.

[0089] 26. The method according to embodiment 23, wherein the cancer is pancreatic cancer, melanoma, breast cancer, lung cancer, head and neck cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma or blood tissue cancer.

[0090] 27. An antibody or antigen-binding fragment according to any one of embodiments 1-19, for treating cancer in a subject, comprising administering the antibody or antigen-binding fragment to the subject.

[0091] 28. An antibody or antigen-binding fragment according to any one of embodiments 1-19, for treating cancer in a subject, comprising one or more inhibitors or antagonists of PD-1, PD-L1, and / or PD-L2.

[0092] 29. The antibody or antigen-binding fragment according to embodiment 28, wherein the one or more inhibitors or antagonists of PD-1, PD-L1, and / or PD-L2 are anti-PD-1 antibodies selected from pembrolizumab, nivolumab, cemiplimab, and pidilizumab.

[0093] 30. The antibody or antigen-binding fragment according to embodiment 28, wherein the one or more inhibitors or antagonists of PD-1, PD-L1, and / or PD-L2 are PD-L1 inhibitors selected from durvalumab, atezolizumab, avelumab, YW243.55.S70, MPDL3280A, MEDI-4736, MSB-0010718C, and MDX-1105.

[0094] 31. The antibody or antigen-binding fragment according to embodiment 27 or 28, wherein the cancer is pancreatic cancer, melanoma, breast cancer, lung cancer, head and neck cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or blood tissue cancer.

[0095] 32. Use of the antibody or antigen-binding fragment according to any one of embodiments 1-19 in the treatment of cancer.

[0096] 33. Use of the antibody or antigen-binding fragment according to any one of embodiments 1-19 in the preparation of a medicament for the treatment of cancer.

[0097] 34. Use of the antibody or antigen-binding fragment according to any one of embodiments 1-19 in the preparation of a medicament for the treatment of cancer, wherein the cancer is pancreatic cancer, melanoma, breast cancer, lung cancer, head and neck cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or blood tissue cancer.

[0098] 35. A nucleic acid molecule encoding the anti-ILT3 antibody or antigen-binding fragment according to any one of embodiments 1-19.

[0099] 36. A vector comprising the nucleic acid molecule according to embodiment 35.

[0100] 37. A host cell comprising the vector according to embodiment 36.

[0101] 38. A method for producing an antibody or antigen-binding fragment according to any one of Embodiments 1-19, comprising:

[0102] (a) providing a host cell comprising the vector of Embodiment 35;

[0103] (b) culturing the host cell in a medium under conditions capable of expressing the antibody or antigen-binding fragment encoded by the vector; and,

[0104] (c) obtaining the antibody or antigen-binding fragment from the medium to provide the antibody or antigen-binding fragment. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1A , Figure 1B , Figure 1C , Figure 1D , Figure 1E and Figure 1F shows a comparison of the selectivity of various of the anti-ILT3 antibodies disclosed herein for monoclonal antibody 9B11 and murine IgG1 (mIgG1), using a cell-based ELISA format. CHO-K1 cells expressing human ILT3 ( Figure 1A ), cynomolgus ILT3 ( Figure 1B ), human ILT5 ( Figure 1C ), human ILT7 ( Figure 1D ), human ILT8 ( Figure 1E ), or human ILT11 ( Figure 1F ) were each tested with monoclonal antibodies p40B5 (LB179.40B5.1A1), p49C6 (LB181.49C6.1A1), and p52B8 (lb181.52B8.1B1); antibody 9B11 (U.S. Patent No. 7,777,008 having the amino acid sequences of SEQ ID NO:33 (light chain) and SEQ ID NO:34 (heavy chain)) and murine IgG1.

[0106] Figure 2A Shows data characteristics of binding affinity, isoelectric point, monomeric species purity, and thermal stability measurements for variants of mAb 10. Terms: "huILT3" refers to human ILT3; "rhILT3" refers to cynomolgus ILT3; "pI" refers to isoelectric point; "Tm" refers to the temperature midpoint of the thermal unfolding curve; "Tagg" refers to the midpoint of the thermal aggregation curve; "SEC" refers to size exclusion ultra performance liquid chromatography.

[0107] Figure 2B Shows mAb 10 (M64V V H1 Relationship between SEC purity and melting temperature of humanized light chain variants of IgG4. V L 1-V L 8 refer to variants having the amino acid sequences shown in SEQ ID NO: 126-133, respectively.

[0108] Figure 3A Show the deuterium-labeled differential thermal map of the amino acid residues of the extracellular domain of human ILT3 bound by the chimeric anti-ILT3 52B8 mouse 52B8 V H Parent / human IgG4(S228P): mouse 52B8 parent V L / human κ antibody ("c58B2"; mAb 73). These hexapeptide domains, which contain the epitopes bound by the antibody (residues 18-23 (ISWGNS; SEQ ID NO: 3), residues 64-69 (IPSMTE; SEQ ID NO: 4), residues 96-101 (MTGAYS; SEQ ID NO: 5), residues 124-131 (QSRSPMDT; SEQ ID NO: 6), residues 152-159 (AQQHQAEF; SEQ ID NO: 7) and residues 184-187 (LLSH; SEQ ID NO: 8)), are located near the boundary of the D1 and D2 domains of the ILT3 extracellular domain. The amino acid sequence of this human extracellular domain with a C-terminal His tag is shown in SEQ ID NO: 1.

[0109] Figure 3B Show the first view and the second view of the surface structure model of the extracellular domain of human ILT3. The dark areas of the model show the positions of the hexapeptide domains containing the epitopes of human ILT3-His bound by c58B8 (mAb 73).

[0110] Figure 3C Is a ribbon diagram showing the positions of the epitopes on the ILT3 extracellular domain: ISWGNS (SEQ ID NO: 3), IPSMTE (SEQ ID NO: 4), MTGAYS (SEQ ID NO: 5), QSRSPMDT (SEQ ID NO: 6), AQQHQAEF (SEQ ID NO: 7) and LLSH (SEQ ID NO: 8).

[0111] Figure 3D Show the deuterium-labeled differential thermal map of the amino acid residues of the extracellular domain of human ILT3 bound by the antibody ZM4.1.

[0112] Figure 3E Show the deuterium-labeled differential thermal map of the amino acid residues of the extracellular domain of human ILT3 bound by the antibody DX446.

[0113] Figure 3F Deuterium-labeled differential thermal map showing amino acid residues of the extracellular domain of human ILT3 bound by antibody DX439.

[0114] Figure 3G Deuterium-labeled differential thermal map showing amino acid residues of the extracellular domain of human ILT3 bound by antibody 9B11.

[0115] Figure 4 Shows the free c52B8 (mAb 73) concentration in blood after multiple doses in humanized tumor models (Panc08.13 and SK-MEL-5). Free c52B8 concentrations are represented by circles and squares. The dashed lines represent simulated historical antibody levels after intravenous bolus administration of 1, 3, 10, or 30 mg / kg of humanized IgG4 in C57BL / 6J mice.

[0116] Figure 5A Shows a human dendritic cell (DC) functional assay demonstrating anti-ILT3 antibody chimeric antibodies, where V from p52B8 fused to IgG4 Fc (c52B8; mAb 73), IgG1 Fc (mAb78), or IgG1(N297A)Fc (mAb 76) H and V L Have comparable ability to activate dendritic cells (DCs). Human immature DCs were prepared and differentiated into CD11c+ dendritic cells with GM-CSF (1000 U / mL) and IL-4 (1000 U / mL) over 5 days. These cells were treated with IL-10, LPS (a Gram-negative bacterial cell wall component and TLR4 ligand (Raetz et al. Ann. Rev. Biochem. 71:635–700 (2002)), and the indicated antibodies at different concentrations for 42 hours. Data shown are the mean and s.d. of two technical replicates. This experiment represents four independent studies. Control IgG had no effect (not shown).

[0117] Figure 5B and Figure 5C Shows that humanized 52B8 (lot 26AVY; mAb 46) is indistinguishable from c52B8 (mAb 73) in a human DC functional assay using DCs from two different healthy human donors. Data shown are the mean and s.d. of two technical replicates. Data shown represent three independent studies using these two donors.

[0118] Figure 6A and Figure 6BIt is shown that the anti-ILT3 antibody c52B8 (mAb 73) and the humanized anti-ILT3 antibody 52B8 (mAb 46; lot 26AVY) reduced the inhibitory capacity of myeloid-derived suppressor cells (MDSC). This T cell inhibition assay was performed with a T cell to MDSC ratio of 4:1. The data shown are the mean and s.d. of three technical replicates at the level of the T cell assay step. The experiments shown represent two independent studies using PBMCs from the same two donors, with qualitatively similar results.

[0119] Figure 7 It is shown that c52B8 inhibited the growth of SK-MEL-5 tumors in SK-MEL-5 human-NSG mice bearing subcutaneous SK-MEL-5 tumors. Animals were randomly assigned to treatment based on tumor volume on day 21 post-transplantation, and 20 mg / kg of c52B8 or isotype control was administered subcutaneously once a week starting on day 21. The data shown in the top graph are the mean and std. error (nine per group). Individual animal tumor growth curves are shown in the middle and bottom graphs. Body weight decreased to a similar extent in both the control group and the 52B8 group. This study represents three independent studies.

[0120] Figure 8A , Figure 8B , Figure 8C and Figure 8D It is shown that c52B8 has an effect in the SK-MEL-5 hu-NSG model in terms of tumor growth and immune activation. Figure 8A It is shown the tumor growth curve. Figure 8B It is shown the CyTOF quantification of TIL collected 7 days after the second dose: CD4 + regulatory T cell %, and the expression level of CD69 on CD4 + T cells; Figure 8C It is shown the sHLA-G level in plasma harvested at the end of the study; Figure 8D It is shown the human CD3 + IHC analysis of T cell infiltration in tumors, 4 tumors per group.

[0121] Figure 9A , Figure 9B , Figure 9C and Figure 9D It is shown the effect of the combination of c52B8 and pembrolizumab in Panc08.13 human-NSG mice. Figure 9A It is shown the tumor growth curve. Figure 9B It is shown the CyTOF quantification of regulatory T cell % and CD69 expression level on CD4+ T cells from tumors harvested at the end of the study; Figure 9C It is shown the plasma sHLA-G level in endpoint blood samples; Figure 9DShow plasma IFNγ and IL-8 levels in end-point blood samples quantified by 10plex MSD (Meso Scale Discovery) for display.

[0122] Figure 10 Show that in the MDSC / T cell inhibition assay, at a T cell to MDSC ratio of 4:1, the humanized anti-ILT3 antibody 52B8 (mAb 46) reduces the inhibitory capacity of MDSCs to a level comparable to that of the chimeric anti-ILT3 antibody c52B8 (mAb 73).

[0123] Figure 11 Show the effect of the combination of the humanized anti-ILT3 antibody 52B8 (mAb 46) and pembrolizumab in the MDSC / T cell inhibition assay at T cell to MDSC ratios of 4:1 or 8:1 using MDSC cells obtained from human donor D001003835.

[0124] Figure 12 Show the effect of the combination of the humanized anti-ILT3 antibody 52B8 (mAb 46) and pembrolizumab in the MDSC / T cell inhibition assay at a T cell to MDSC ratio of 8:1 using MDSC cells obtained from human donor D001003180.

[0125] Figure 13 Show the effect of the combination of the humanized anti-ILT3 antibody 52B8 (mAb 46) and pembrolizumab in the MDSC / T cell inhibition assay at a T cell to MDSC ratio of 4:1 using MDSC cells obtained from human donor D001003507.

[0126] Figure 14 Show the effect of the combination of the humanized anti-ILT3 antibody 52B8 (mAb 46) and pembrolizumab in the MDSC / T cell inhibition assay at a T cell to MDSC ratio of 8:1 using MDSC cells obtained from human donor D001003428.

[0127] Figure 15 Show the effect of the combination of the humanized anti-ILT3 antibody 52B8 (mAb46) and pembrolizumab in the mixed lymphocyte reaction of IL-10 polarized human monocyte-derived dendritic cells with allogeneic CD8+ T cells, measuring interferon γ (IFNγ) in the culture supernatant as a readout of T cell activation after four days of incubation. Detailed Description

[0128] The present invention provides non-confounding monoclonal antibodies specific for the inhibitory receptor immunoglobulin-like transcript 3 (ILT3) expressed on the surface of myeloid immune cells.

[0129] Definition

[0130] “Immunoglobulin-like transcript 3” (abbreviated herein as “ILT3” and also known as LIR-5, LILRB4, or CD85k), as used herein and unless otherwise specified, refers to the human member of the ILT3 family that is selectively expressed by myeloid antigen-presenting cells (APCs) such as monocytes, macrophages, and dendritic cells (e.g., monocyte-derived dendritic cells differentiated in the presence of IL-10 or vitamin D 3 ).

[0131] As used herein, “antibody” refers to intact immunoglobulins, including recombinantly produced forms, and includes any form of an antibody that exhibits the desired biological activity. Thus, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, diabodies, humanized camelid heavy-chain antibodies, and non-human / human chimeric antibodies. A “parent antibody” is an antibody obtained by exposing the immune system to an antigen prior to modification of the antibody for its intended use, such as the humanization of a non-human antibody for use as a human therapeutic antibody.

[0132] In one embodiment, “antibody” refers to a glycoprotein comprising at least two heavy chains (HCs) and two light chains (LCs) interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain consists of a heavy-chain variable region or domain (abbreviated herein as V H ) and a heavy-chain constant region or domain. In certain naturally occurring IgG, IgD, and IgA antibodies, the heavy-chain constant region consists of three domains, CH1, CH2, and CH3. In certain naturally occurring antibodies, each light chain consists of a light-chain variable region or domain (abbreviated herein as V L ) and a light-chain constant region or domain. The light-chain constant region consists of one domain, CL. Human V H includes six family members: V H 1, V H 2, V H 3, V H 4, V H 5, and V H 6, and human V L family includes 16 family members: V κ 1, V κ 2, V κ 3, V κ 4, V κ 5, V κ 6, V λ 1, V λ 2,, Vλ 3, V λ 4, V λ 5, V λ 6, V λ 7, V λ 8, V λ 9, and V λ 10. Each of these family members can be further divided into specific subtypes.

[0133] V can be H and V L regions can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), which are interspersed with more conserved regions, called framework regions (FRs). Each V H and V L consists of three CDR regions and four FR regions, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). The assignment of amino acids to each domain is usually based on the following definitions: Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5 th ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883.

[0134] Generally, although an antibody contains six CDRs, three on V H and three on V LThe above three, but the prior art has recognized that in most cases, the CDR3 region of the heavy chain is the main determinant of antibody specificity, and examples of the generation of specific antibodies based solely on the specificity of the CDR3 of the heavy chain are known in the art (e.g., Beiboer et al., J. Mol. Biol. 296:833-849 (2000); Klimka et al., British J. Cancer 83:252-260 (2000); Rader et al., Proc. Natl. Acad. Sci. USA 95:8910-8915 (1998); Xu et al., Immunity 13:37-45 (2000). See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (defining the CDR regions of antibodies according to the sequences); also see Chothia and Lesk (1987) J. Mol. Biol. 196:901-917 (defining the CDR regions of antibodies according to the structures)).

[0135] The following general rules shown in Table 1 can be used to determine the CDRs in an antibody sequence. There are rare examples where these almost invariant features do not occur; however, the Cys residue is the most conserved feature.

[0136]

[0137]

[0138] Generally, the basic antibody structural unit comprises a tetramer. Each tetramer includes two pairs of identical polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50 - 70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids, which is mainly responsible for antigen recognition. The carboxyl-terminal portion of the heavy chain can define a constant region, which is mainly responsible for the effector functions of the antibody. Generally, human light chains are divided into κ and λ light chains. In addition, human heavy chains are usually classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. In the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 amino acids. See generally Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989).

[0139] The heavy chain of an antibody may or may not contain a terminal lysine (K) residue, or a terminal glycine and lysine (GK) residue. Thus, in certain embodiments of the anti-ILT3 antibodies herein, it includes the amino acid sequence of the heavy chain constant region shown herein that lacks the terminal lysine but terminates with a glycine residue, and further includes embodiments in which the terminal glycine residue is also lacking. This is because the terminal lysine and sometimes glycine and lysine together can be cleaved during antibody expression or when introduced into the human body, without an obvious adverse effect on antibody efficacy, stability or immunogenicity. In some cases, the nucleic acid molecule encoding the heavy chain may deliberately omit the codon encoding the terminal lysine or the codons encoding the terminal lysine and glycine.

[0140] As used herein, "antigen-binding fragment" refers to a fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to an antigen bound by the full-length antibody, such as a fragment that retains one or more CDR regions. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab') 2 and Fv fragments; diabodies; single-chain antibody molecules, such as scFv; nanobodies and multispecific antibodies formed from antibody fragments.

[0141] As used herein, "Fab fragment" comprises one light chain and C H 1 and the variable region of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A "Fab fragment" can be the product of papain digestion of an antibody.

[0142] As used herein, "Fab' fragment" comprises one light chain and a portion or fragment of one heavy chain, which contains V HThe region between the domain and the CH1 domain, and between the CH1 and CH2 domains, allows for the formation of inter-chain disulfide bonds between the two heavy chains of two Fab' fragments to form F(ab') 2 molecule.

[0143] As used herein, "F(ab') 2 fragment" comprises two light chains and two heavy chains, said two light chains and two heavy chains comprising V H domains and a portion of the constant region between the CH1 and CH2 domains such that inter-chain disulfide bonds are formed between the two heavy chains. Thus, the F(ab') 2 fragment consists of two Fab' fragments that are joined together by a disulfide bond between the two heavy chains. "F(ab') 2 fragment" can be the product of pepsin digestion of an antibody.

[0144] As used herein, the "Fv region" includes the variable regions from both the heavy and light chains, but lacks the constant regions.

[0145] These and other potential construction methods are described in Chan & Carter (2010) Nat. Rev. Immunol. 10:301. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the utility of the fragments is screened in the same manner as for intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0146] As used herein, the "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

[0147] As used herein, a "diabody" refers to a small antibody fragment having two antigen-binding sites that comprises a heavy chain variable domain (V L ) linked to a light chain variable domain in the same polypeptide chain (V H -V L or V L -V H)。By using linkers that are too short to allow pairing between the two domains on the same chain, these domains are forced to pair with the complementary domains on the other chain and generate two antigen-binding sites. Bispecific antibodies are more fully described, for example, in EP 404,097; WO 93 / 11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. For a review of engineered antibody variants, see generally Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

[0148] As used herein, a "bispecific antibody" is an artificial hybrid antibody that has two different heavy / light chain pairs and thus has two different binding sites. For example, a bispecific antibody can comprise a first heavy / light chain pair that comprises one heavy chain and one light chain of a first antibody that comprises at least six CDRs of the anti-ILT3 antibodies disclosed herein or an embodiment in which one or more of the six CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, and a second heavy / light chain pair that comprises one heavy chain and one light chain of a second antibody that is specific for an antigen of interest other than ILT3. Bispecific antibodies can be generated by a variety of methods, including fusion of hybridomas or ligation of Fab' fragments. See, for example, Songsivilai, et al., (1990) Clin. Exp. Immunol. 79:315-321, Kostelny, et al., (1992) J Immunol. 148:1547-1553. Additionally, bispecific antibodies can be formed as "bispecific antibodies" (Holliger, et al., (1993) PNAS USA 90:6444-6448) or "Janusins" (Traunecker, et al., (1991) EMBO J. 10:3655-3659 and Traunecker, et al., (1992) Int. J. Cancer Suppl. 7:51-52).

[0149] As used herein, an "isolated" antibody or antigen-binding fragment thereof is at least partially free of other biomolecules from the cells or cell culture in which they are produced, such biomolecules including nucleic acids, proteins, lipids, carbohydrates, or other materials such as cell debris and growth medium. An isolated antibody or antigen-binding fragment may further be at least partially free of expression system components, such as biomolecules from a host cell or its growth medium. Generally, the term "isolated" is not intended to mean the complete absence of such biomolecules or the absence of water, buffers, or salts, nor the components of a pharmaceutical formulation containing the antibody or fragment.

[0150] As used herein, a "monoclonal antibody" refers to a population of antibodies that are substantially homogeneous, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally-occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a multiplicity of different antibodies having different amino acid sequences in their variable domains that are usually specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies useful in the present invention may be prepared by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or may be prepared by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al. (1991) Nature 352:624-628 and Marks et al. (1991) J. Mol. Biol. 222:581-597. See also Presta (2005) J. Allergy Clin. Immunol. 116:731.

[0151] As used herein, a "chimeric antibody" is an antibody having variable domains from a first antibody and constant domains from a second antibody, where (i) the first and second antibodies are from different species (U.S. Pat. No. 4,816,567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855) or (ii) the first and second antibodies are from different isotypes, e.g., variable domains from an IgG1 antibody and constant domains from an IgG4 antibody. In one aspect, the variable domains are obtained from a non-human antibody, such as a murine antibody ("parent antibody"), and the constant domain sequences are obtained from a human antibody. In another aspect, the variable domains are humanized variable domains from a murine antibody and the constant domains from a human antibody.

[0152] As used herein, a "humanized antibody" refers to an antibody form that contains sequences from both human and non-human (e.g., murine, rat) antibodies. Generally, a humanized antibody will contain all of at least one and usually two variable domains, where the hypervariable loops correspond to those of the non-human immunoglobulin, and all or substantially all of the framework (FR) regions are sequences of those human immunoglobulin sequences. A humanized antibody may optionally contain at least a portion of a human immunoglobulin constant region (Fc).

[0153] "Humanization" (also referred to as "reshaping" or "CDR grafting") has now become a well-established technique for reducing the immunogenicity of monoclonal antibodies (mAbs) of xenogeneic origin (usually rodents) and improving effector functions (ADCC, complement activation, C1q binding). Engineering of mAbs using molecular biology techniques, however, often results in loss of the binding affinity and / or specificity of the original mAb when simply grafting rodent complementarity determining regions (CDRs) into a human framework. To humanize an antibody, the design of a humanized antibody includes variants such as conservative amino acid substitutions in CDR residues, as well as back substitutions (back mutations) from rodent mAb residues into the human framework regions. Positions can be identified or recognized by sequence comparison for structural analysis or by homology modeling of the 3D structure of the variable region. The process of affinity maturation has recently used phage libraries to vary the amino acids at selected positions. Similarly, many methods have been used to select the most suitable human framework for grafting rodent CDRs. As the dataset of known parameters of antibody structures increases, the refinement and sophistication of these techniques also increase. Consensus sequences or germline sequences from a single antibody, or fragments of framework sequences within each light or heavy chain variable region from several different human monoclonal antibodies can be used. Another approach to humanization is to modify the surface residues of a rodent sequence with only the most common residues found in human monoclonal antibodies, a method known as "resurfacing" or "veneering". Human or humanized antibodies are generally substantially non-immunogenic in humans.

[0154] As used herein, a "non-human amino acid sequence" with respect to an antibody or immunoglobulin refers to an amino acid sequence that is characteristic of the amino acid sequences of non-human mammals. The term does not include the amino acid sequences of antibodies or immunoglobulins obtained from fully human antibody libraries where the diversity in the library is generated by computer (see, e.g., U.S. Patent Nos. 8,877,688 or 8,691,730).

[0155] As used herein, "effector function" refers to those biological activities that are attributable to the Fc region of an antibody and that vary with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0156] As used herein, "conservative modified variants" or "conservative substitutions" refer to the replacement of an amino acid with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation, and rigidity, etc.) such that changes can often be made without altering the biological activity of the protein. Those skilled in the art recognize that, generally, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.)). Additionally, substitutions of amino acids that are structurally or functionally similar are not likely to disrupt biological activity. Exemplary conservative substitutions are set forth in Table 2.

[0157]

[0158]

[0159] As used herein, the term "epitope" or "antigenic determinant" refers to a site on an antigen (such as ILT3) to which an immunoglobulin or antibody specifically binds. Epitopes within a protein antigen can be formed either by contiguous amino acids (usually linear epitopes) or by non-contiguous amino acids juxtaposed by the tertiary folding of the protein (usually conformational epitopes). Epitopes formed by contiguous amino acids generally but not always remain upon exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents. Contiguous linear epitopes comprise a peptide domain on the antigen that contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. Non-contiguous conformational epitopes comprise one or more peptide domains or regions on the antigen that are bound by an antibody and are interspersed with one or more amino acids or peptide domains that are not bound by that antibody, each domain independently containing at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. Methods for determining which epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, in which the reactivity of overlapping or contiguous peptides (such as from ILT3) with a given antibody (such as an anti-ILT3 antibody) is tested. Methods for determining the spatial conformation of an epitope include techniques known in the art and described herein, such as X-ray crystallography, two-dimensional nuclear magnetic resonance, and HDX-MS (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996)).

[0160] The term "epitope mapping" refers to the process of identifying the molecular determinants on an antigen involved in antibody-antigen recognition using techniques known in the art and described herein, such as X-ray crystallography, two-dimensional nuclear magnetic resonance, and hydrogen deuterium exchange mass spectrometry (HDX-MS).

[0161] The term "bind the same epitope" in relation to two or more antibodies refers to the combination of antibodies that bind the same stretch of amino acid residues or amino acid fragments, as determined by a given method. Techniques for determining whether an antibody binds the "same epitope on ILT3" as an antibody described herein include epitope mapping methods such as X-ray analysis of crystals of the antigen:antibody complex, which provides atomic resolution of the epitope, and HDX-MS. Other methods that monitor the binding of an antibody to an antigen fragment (e.g., a proteolytic fragment) or an antigen mutant variant, where loss of binding is caused by modification of an amino acid residue within the antigen sequence, are generally considered indicative of epitope components (e.g., alanine-scanning mutagenesis - Cunningham & Wells (1985) Science 244:1081). Additionally, computational combinatorial methods for epitope mapping can be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from a combinatorial phage display peptide library.

[0162] An antibody that "competes with another antibody for binding to a target, e.g., ILT3" is an antibody that inhibits (partially or completely) the binding of another antibody to the target, i.e., ILT3. Known competition assays can be used to determine whether two antibodies compete with each other for binding to the target, i.e., whether one antibody inhibits and to what extent the binding of another antibody to the target. In certain embodiments, the antibody competes with and inhibits the binding of another antibody to the target by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. The level of inhibition or competition may vary depending on which antibody is the "blocking antibody" (i.e., the cold antibody that is incubated with the target first). Competition assays can be described, for example, in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi:10.1101 / pdb.prot4277 or in Chapter 11 of "Using Antibodies" by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA 1999. Competitive antibodies bind the same epitope, overlapping epitopes, or adjacent epitopes (e.g., demonstrated by steric hindrance).

[0163] Other competitive binding assays include: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al., Methods in Enzymology 9:242 (1983)); and the like. Solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phase direct labeling assay, solid phase direct labeling sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct labeling RIA using 1-125 labeling (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and direct labeling RIA (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

[0164] As used herein, "specific binding" with respect to an antigen or molecule such as human ILT3 refers to the preferential binding of an antibody or other ligand, in whole or in part, to human ILT3 rather than to other molecules, particularly molecules found in human blood or serum. Antibodies typically bind specifically to their cognate antigen with high affinity, which can be demonstrated by 10 -7 Up to 10 -11 M or less dissociation constant (K D ) reflects that any value greater than 10 -6 M of K D It is usually considered to represent non-specific binding. As used herein, an antibody that "specifically binds" or "specifically binds" to human ILT3 refers to an antibody that binds to human ILT3 with high affinity, meaning that K D For 10 -7 M or less, in a specific embodiment, K D For 10 -8 M or smaller, or 5×10 -9 M or smaller, or 10 -8 M to 10 -11 M or less, but does not bind with measurable binding to closely related proteins such as human ILT5, human ILT7, human ILT8, and human ILT11 as determined in cell ELISA or Biacore analysis using 10 μg / mL antibody.

[0165] As used herein, an antigen is "substantially identical" to a given antigen if it exhibits a high degree of amino acid sequence identity to the given antigen, e.g., if it exhibits at least 80%, at least 90%, at least 90%, 95%, at least 97% or at least 99% or greater amino acid sequence identity to the amino acid sequence of the given antigen. By way of example, an antibody that specifically binds human ILT3 may also cross-react with ILT3 from certain non-human primate species (e.g., macaque or cynomolgus monkey).

[0166] As used herein, an "isolated nucleic acid molecule" refers to DNA or RNA or some combination thereof of genomic, mRNA, cDNA or synthetic origin, which is unassociated with all or a portion of the polynucleotide with which the isolated polynucleotide is found in nature. Or joined to polynucleotides that are not linked in nature. For purposes of this disclosure, it should be understood that a nucleic acid molecule that "comprises" a particular nucleotide sequence does not include an entire chromosome. In addition to the particular sequence, an isolated nucleic acid molecule that "comprises" a particular nucleic acid sequence may include up to ten or even up to twenty or more other protein or portions or fragments thereof encoding sequences, or may include operably linked regulatory sequences that control the expression of the coding region of the recited nucleic acid sequence, and / or may include vector sequences.

[0167] As used herein, "treatment" refers to the administration of a therapeutic agent, e.g., a composition comprising any antibody or antigen-binding fragment thereof of the invention, to a subject or patient suffering from one or more diseases, to a symptom or suspected disease for which the agent has therapeutic or prophylactic activity. Generally, the agent is administered in an amount effective to alleviate one or more symptoms of the disease in the subject or population being treated, whether by inducing regression of such symptoms to any clinically measurable extent or by inhibiting their development. The amount of a therapeutic agent effective to alleviate any particular disease symptom may vary depending on factors such as the disease state, the age and weight of the patient, and the ability of the agent to elicit a desired response in the subject. Whether a disease symptom has been alleviated can be evaluated by any clinical measurement commonly used by a physician or other skilled healthcare provider to assess the severity or progression status of the symptom. The term further includes delaying the development of symptoms associated with a disorder and / or alleviating the severity of the symptoms of the disorder. The term also includes ameliorating existing uncontrolled or unwanted symptoms, preventing other symptoms, and ameliorating or preventing the underlying cause of such symptoms. Thus, the term indicates that a beneficial result has been conferred on a human or animal subject suffering from a disorder, disease or symptom or having the potential to develop such a disorder, disease or symptom.

[0168] As used herein, "treatment", as it applies to human or veterinary subjects, refers to both therapeutic treatment and diagnostic applications. "Treatment", when applied to a human or veterinary subject, encompasses bringing an antibody or antigen-binding fragment of the invention into contact with the human or animal subject.

[0169] As used herein, "therapeutically effective amount" refers to the amount of a particular substance sufficient to achieve the desired effect in a subject being treated. For example, this can be the amount necessary to inhibit ILT3 activation when co-administered with pembrolizumab or the amount necessary to enhance pembrolizumab responsiveness.

[0170] As used herein, the term "PD-1" refers to the programmed death 1 (PD-1) protein, which is an inhibitory member of the extended CD28 / CTLA-4 family of T cell regulators (Okazaki et al. (2002) Curr Opin Immunol 14:391779-82; Bennett et al. (2003) J. Immunol. 170:711-8). Other members of the CD28 family include CD28, CTLA-4, ICOS, and BTLA. The PD-1 gene encodes a 55 kDa type I transmembrane protein (Agata et al., (1996) Int Immunol. 8:765-72). Two ligands of PD-1, PD-L1 (B7-H1) and PD-L2 (B7-DC), have been identified, which have been shown to downregulate T cell activation upon binding to PD-1 (Freeman et al. (2000(J. Exp. Med. 192:1027-34; Carter et al. (2002) Eur. J. Immunol. 32:634-43). PD-1 is referred to as an immunosuppressive protein that negatively regulates TCR signaling (Ishida, Y. et al. (1992) EMBO J. 11:3887-3895; Blank, C. et al. (Epub Dec 29, 2006). Immunol. Immunother. 56(5):739-745). The interaction between PD-1 and PD-L1 can act as an immune checkpoint, which can, for example, result in reduced tumor-infiltrating lymphocytes, reduced T cell receptor-mediated proliferation, and / or immune escape of cancer cells (Dong et al. (2003) J. Mol. Med. 81:281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314; Konishi et al. (2004) Clin. Cancer Res. 10:5094-100). The immunosuppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1 or PD-L2. This effect is additive when the interaction between PD-1 and PD-L2 is also blocked (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99:12293-7; Brown et al. (2003J. Immunol. 170:1257-66).

[0171] Antibodies and antigen-binding fragments

[0172] The present invention provides isolated chimeric, humanized and human antibodies and antigen-binding fragments that specifically bind to ILT3 and have no measurable binding to closely related proteins (e.g., ILT5, ILT7, ILT8, and ILT11), as determined using 10 μg / mL antibody in a cell ELISA or Biacore assay. The anti-ILT3 antibodies increase the activity of antigen-presenting cells and dendritic cells, reduce the activity of monocyte repressors, and increase T cell priming. Accordingly, the present invention also includes the use of anti-ILT3 antibodies in the treatment of cancer in monotherapy and in combination with anti-PD-1 or anti-PD-L1 antibodies for the treatment of cancer in first-line, second-line, or third-line therapy.

[0173] Anti-ILT3 antibodies include any antibody having an amino acid sequence disclosed herein, and include any antibody that comprises (i) at least one, two, three, four, five, or six CDRs of an antibody having an amino acid sequence disclosed herein; or (ii) although not having the CDR amino acid sequences disclosed herein, binds to the same epitope on ILT3 as the antibodies disclosed in the present invention and can modulate ILT3 receptor signaling such that the antibody increases the activity of antigen-presenting cells and dendritic cells, reduces the activity of inhibitory monocytes, and increases the ability to prime T cells. In a specific aspect, the antibody has no measurable binding to human ILT5, human ILT7, human ILT8, and human ILT11, as determined using 10 μg / mL antibody in a cell ELISA or in a Biacore assay. The term specifically excludes antibodies that comprise at least one CDR of the following antibodies: antibody ZM4.1 or antibody 9B11 or U.S. Patent Nos. 7,777,008 and 8,901,281 or U.S. Patent Application Publication Nos. 20090202544, 20150110714, 20150139986, and 20170267759; and any other antibodies disclosed in International Publication Nos. WO2013043569, WO2013181438, WO2014116846, WO2016049641, WO2016127427, WO2018089300, and WO2018148494.

[0174] An anti-ILT3 antigen-binding fragment, etc., includes any protein or peptide comprising a molecule that comprises (i) at least a portion of an anti-ILT3 antibody disclosed herein according to an amino acid sequence, (ii) at least one, two, three, four, five, or six CDRs of an antibody disclosed herein according to an amino acid sequence, or (iii) although not having the CDR amino acid sequences disclosed herein, but binds to the same epitope on ILT3 as the antibody disclosed in the present invention and can modulate ILT3 receptor signaling such that the antigen-binding fragment increases the activity of antigen-presenting cells and dendritic cells, reduces the activity of inhibitory monocytes, and increases the priming ability of T cells. In a specific aspect, the antigen-binding fragment does not have measurable binding to human ILT5, human ILT7, human ILT8, and human ILT11, as determined using 10 μg / mL of the anti-ILT3 antigen-binding fragment in a cell ELISA or in a Biacore assay. The term specifically excludes antigen-binding fragments comprising at least one CDR of the following antibodies: antibody ZM4.1 or antibody 9B11 or U.S. Patent Nos. 7,777,008 and 8,901,281 or U.S. Patent Application Publication Nos. 20090202544, 20150110714, 20150139986, and 20170267759; and any other antibodies disclosed in International Publications WO2013043569, WO2013181438, WO2014116846, WO2016049641, WO2016127427, WO2018089300, and WO2018148494.

[0175] In a further embodiment, the anti-ILT3 antibody comprises any antibody that comprises (i) at least the HC-CDR3 of an antibody disclosed herein according to the amino acid sequence; or (ii) although not having the HC-CDR3 amino acid sequence disclosed herein, binds to the same epitope on ILT3 as the antibody disclosed in the present invention and can modulate ILT3 receptor signaling such that the antibody increases the activity of antigen-presenting cells and dendritic cells, reduces the activity of inhibitory monocytes, and increases the priming ability of T cells. In a specific aspect, the antibody does not have measurable binding to human ILT5, human ILT7, human ILT8, and human ILT11, as determined using 10 μg / mL antibody in a cell ELISA or in a Biacore assay. The term specifically excludes antibodies that comprise at least one CDR of the following antibodies: antibody ZM4.1 or antibody 9B11 or U.S. Patent Nos. 7,777,008 and 8,901,281 or U.S. Patent Application Publication Nos. 20090202544, 20150110714, 20150139986, and 20170267759; and any other antibodies disclosed in International Publications WO2013043569, WO2013181438, WO2014116846, WO2016049641, WO2016127427, WO2018089300, and WO2018148494.

[0176] An anti-ILT3 antigen-binding fragment, etc., includes any protein or peptide comprising a molecule that comprises (i) at least a portion of an anti-ILT3 antibody as disclosed herein according to an amino acid sequence, (ii) at least the HC-CDR3 of an antibody as disclosed herein according to an amino acid sequence, or (iii) although not having the HC-CDR3 amino acid sequence as disclosed herein, but binds to the same epitope on ILT3 as the antibody disclosed in the present invention and can modulate ILT3 receptor signaling such that the antigen-binding fragment increases the activity of antigen-presenting cells and dendritic cells, reduces the activity of inhibitory monocytes, and increases the priming ability of T cells. In a specific aspect, the antigen-binding fragment does not have a measurable binding to human ILT5, human ILT7, human ILT8, and human ILT11, as determined using 10 μg / mL of the anti-ILT3 antigen-binding fragment in a cell ELISA or in a Biacore assay. The term specifically excludes antigen-binding fragments comprising at least one CDR of the following antibodies: antibody ZM4.1 or antibody 9B11 or U.S. Patent Nos. 7,777,008 and 8,901,281 or U.S. Patent Application Publication Nos. 20090202544, 20150110714, 20150139986, and 20170267759; and any other antibodies disclosed in International Publications WO2013043569, WO2013181438, WO2014116846, WO2016049641, WO2016127427, WO2018089300, and WO2018148494.

[0177] In certain embodiments, the anti-ILT3 antibody is a human or humanized anti-ILT3 antibody or antigen-binding fragment or a chimeric anti-ILT3 antibody or antigen-binding fragment that comprises the HC-CDR3 of the anti-ILT3 antibody molecule disclosed herein or the H3-CDR3 as shown in Table 3.

[0178] In certain embodiments, the anti-ILT3 antibody is a human or humanized anti-ILT3 antibody or antigen-binding fragment or a chimeric anti-ILT3 antibody or antigen-binding fragment that comprises the HC-CDR1, HC-CDR2, HC-CDR3, LC-CDR1, LC-CDR2, and LC-CDR3 as disclosed herein or in Table 3.

[0179]

[0180] In certain embodiments, the anti-ILT3 antibody is a human or humanized anti-ILT3 antibody or antigen-binding fragment or a chimeric anti-ILT3 antibody or antigen-binding fragment, each of which comprises a heavy-chain variable domain (V) having a heavy-chain complementarity-determining region (HC-CDR) 3 H), which comprises an amino acid sequence selected from SEQ ID NO: 22, 49, 57, 65, 73, 81, 89, 97, and 105, or an amino acid sequence having 3, 2, or 1 difference from an amino acid sequence selected from SEQ ID NO: 22, 49, 57, 65, 73, 81, 89, 97, and 105. In another embodiment, the antibody or antigen-binding fragment binds to an epitope on human ILT3, wherein the epitope comprises at least one amino acid selected from one or more of the amino acid sequences of SEQ ID NO: 3, 4, 5, 6, 7, and 8. In another embodiment, the antibody or antigen-binding fragment binds to an epitope on human ILT3, wherein the epitope comprises the amino acid sequences shown in SEQ ID NO: 3, 4, 5, 6, 7, and 8. In a particular embodiment, the amino acid sequence difference is a conservative change / substitution.

[0181] In a particular embodiment, the anti-ILT3 antibody is a humanized or chimeric anti-ILT3 antibody disclosed herein. In a particular embodiment, the anti-ILT3 antibody is a human or humanized anti-ILT3 antibody or antigen-binding fragment or a chimeric anti-ILT3 antibody or antigen-binding fragment that binds to the same epitope bound by the anti-ILT3 antibody disclosed herein or competes with the anti-ILT3 antibody disclosed herein for binding, and the antibody comprises fewer than three CDRs of the anti-ILT3 antibody disclosed herein or no CDRs of the anti-ILT3 antibody disclosed herein.

[0182] The present invention further provides an antibody or an antigen-binding fragment thereof, which comprises (i) at least six complementarity-determining regions (CDRs) of an anti-immunoglobulin-like transcript 3 (ILT3) antibody; or (ii) at least six CDRs of an anti-ILT3 antibody, wherein one or more of the six CDRs have one, two or three amino acid substitutions, additions, deletions or combinations; wherein the six CDRs of the anti-ILT3 antibody comprise a heavy chain (HC)-CDR1 having the amino acid sequence shown in SEQ ID NO: 17, 47, 55, 63, 71, 79, 87, 95 or 103; an HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 18, 48, 56, 64, 72, 80, 88, 96 or 104; an HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 22, 49, 57, 65, 73, 81, 89, 97 or 105; a light chain (LC)-CDR1 having the amino acid sequence shown in SEQ ID NO: 27, 50, 58, 66, 74, 82, 90, 98 or 106; an LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 43, 51, 59, 67, 75, 83, 91, 99 or 107; and an LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 44, 60, 68, 76, 84, 92, 100 or 108; and wherein the antibody or antigen-binding fragment specifically binds to human or macaque ILT3 or both human and macaque ILT3. In certain embodiments, the amino acid sequence differences are conservative changes / substitutions.

[0183] In certain embodiments, the present invention provides an antibody or an antigen-binding fragment thereof, which comprises six CDRs of an anti-ILT3 antibody, the anti-ILT3 antibody comprising a heavy chain (HC)-CDR1 having the amino acid sequence shown in SEQ ID NO: 17; an HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 19, 20 or 21; an HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 23, 24, 25 or 26; a light chain (LC)-CDR1 having the amino acid sequence shown in SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, 41 or 42; an LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 43; and an LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 44.

[0184] In certain embodiments, the present invention provides an antibody or antigen-binding fragment thereof comprising the six CDRs of an anti-ILT3 antibody, said anti-ILT3 antibody having a heavy chain (HC)-CDR1 with the amino acid sequence shown in SEQ ID NO: 17; an HC-CDR2 with the amino acid sequence shown in SEQ ID NO: 20; an HC-CDR3 with the amino acid sequence shown in SEQ ID NO: 23; a light chain (LC)-CDR1 with the amino acid sequence shown in SEQ ID NO: 41; an LC-CDR2 with the amino acid sequence shown in SEQ ID NO: 43; and an LC-CDR3 with the amino acid sequence shown in SEQ ID NO: 44.

[0185] In certain embodiments, the present invention provides the above antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises a heavy chain variable domain (V H ), which has a framework selected from: human V H 1, V H 2, V H 3, V H 4, V H 5, and V H 6 families, and variants thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof; and (b) a light chain variable domain (V L ), which has a framework selected from: human V κ 1, V κ 2, V κ 3, V κ 4, V κ 5, V κ 6, V λ 1, V λ 2,, V λ 3, V λ 4, V λ 5, V λ 6, V λ 7, V λ 8, V λ 9, and V λ 10 families, and variants thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

[0186] In certain embodiments, the present invention provides the above-described antibody or antigen-binding fragment, wherein the antibody comprises a human IgG1, IgG2, IgG3, or IgG4 heavy chain (HC) constant domain or a variant thereof, which comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof as compared to the amino acid sequence of the native IgG1, IgG2, IgG3, or IgG4 isotype.

[0187] In certain embodiments, the present invention provides the above-described antibody or antigen-binding fragment, wherein the antibody comprises a human κ or λ light chain constant domain or a variant thereof, which comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid substitutions, additions, deletions, or combinations thereof as compared to the amino acid sequence of the native human κ or λ light chain domain.

[0188] In a specific embodiment, the present invention provides the above-described antibody or antigen-binding fragment, wherein the antibody comprises (i) a human heavy chain variable domain (V H ), which has a framework selected from the human V H 3 family, and a human light chain variable domain (V L ), which has a framework selected from the human V κ 1, V κ 3, and V κ 4 families; (ii) a human IgG1 or IgG4 heavy chain (HC) constant domain or a variant thereof that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof as compared to the amino acid sequence of the native IgG1 or IgG4 isotype; (iii) a human κ or λ light chain constant domain or a variant thereof that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof as compared to the amino acid sequence of the native human κ or λ light chain domain. In certain embodiments, the amino acid sequence differences are conservative changes / substitutions.

[0189] In certain embodiments, the present invention provides the above-described antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises a heavy chain variable domain (V H ) and a light chain variable domain (V L):Specifically, SEQ ID NO:15 and SEQ ID NO:16; SEQ ID NO:45 and SEQ ID NO:46; SEQ ID NO:53 and SEQ ID NO:54; SEQ ID NO:61 and SEQ ID NO:62; SEQ ID NO:69 and SEQ ID NO:70; SEQ ID NO:77 and SEQ ID NO:78; specifically, SEQ ID NO:85 and SEQ ID NO:86; SEQ ID NO:93 and SEQ ID NO:94; SEQ ID NO:101 and SEQ ID NO:102.

[0190] In certain embodiments, the present invention provides the above-mentioned antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises a heavy chain variable domain (V) having the amino acid sequence shown in SEQ ID NO: 117, 118, 119, 120, 121, 122, 123, 124 or 125 H ) and a light chain variable domain (V) having the amino acid sequence shown in SEQ ID NO: 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140 or 141. L )

[0191] In certain embodiments, the present invention provides the above-mentioned antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises a heavy chain variable domain (V) having the amino acid sequence shown in SEQ ID NO: 118 H ) and a light chain variable domain (V) having the amino acid sequence shown in SEQ ID NO: 140. L )

[0192] In certain embodiments, the present invention provides the above-mentioned antibody or antigen-binding fragment, wherein the antibody comprises a heavy chain (HC) constant domain, and the constant domain comprises the amino acid sequence shown in SEQ ID NO: 9, 10, 11, 12 or 13 and variants of SEQ ID NO: 9, 11, 12 or 13, wherein the HC lacks a C-terminal lysine or glycine-lysine.

[0193] In certain embodiments, the present invention provides the above-mentioned antibody or antigen-binding fragment, wherein the antibody comprises a light chain (LC) constant domain, and the light chain constant domain comprises the amino acid sequence shown in SEQ ID NO: 14.

[0194] In certain embodiments, the present invention provides the above-mentioned antibody or antigen-binding fragment, wherein the antibody comprises a heavy chain (HC), the heavy chain comprising the amino acid sequence of SEQ ID NO: 142, 143, 144, 148, 149, 150, 167, 168, 169, 170, 174, 175, 176, 177, 178, 182, 183, 184, 185, 186, 187, 191, 192 or 193, and a variant of the HC comprising the amino acid sequence of SEQ ID NO: 143, 144, 148, 149, 150, 167, 168, 169, 170, 174, or 175, wherein the HC lacks a C-terminal lysine or glycine-lysine.

[0195] In certain embodiments, the present invention provides the above-mentioned antibody or antigen-binding fragment, wherein the antibody comprises a light chain (LC), the light chain (LC) comprising the amino acid sequence shown in SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165 or 166.

[0196] In certain embodiments, the present invention provides the above-mentioned antibody or antigen-binding fragment, wherein the antibody comprises a heavy chain (HC), the heavy chain comprising the amino acid sequence of SEQ ID NO: 142, 143, 144, 148, 149, 150, 167, 168, 169, 170, 174, 175, 176, 177, 178, 182, 183, 184, 185, 186, 187, 191, 192 or 193; and a light chain (LC), the light chain comprising the amino acid sequence shown in SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165 or 166, and a variant of the HC comprising the amino acid sequence of SEQ ID NO: 143, 144, 148, 148, 149, 150, 167, 168, 169, 170, 174 or 175, wherein the HC lacks a C-terminal lysine or glycine-lysine.

[0197] In certain embodiments, the present invention provides an antibody selected from the antibodies shown in Table 4.

[0198] In certain embodiments, the present invention provides the above-mentioned antibody or antigen-binding fragment, wherein the antibody comprises a heavy chain (HC) having the amino acid sequence of SEQ ID NO: 143 and a light chain (LC) having the amino acid sequence of SEQ ID NO: 165, and a variant wherein the HC lacks a C-terminal lysine or glycine-lysine.

[0199] In certain embodiments, the present invention provides the above-mentioned antibody or antigen-binding fragment, wherein the antibody comprises a human IgG1, IgG2, IgG3 or IgG4 heavy chain (HC) constant domain or a variant thereof that contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native IgG1, IgG2, IgG3 or IgG4 isotype, and variants thereof in which the HC lacks a terminal lysine or glycine-lysine.

[0200] In some embodiments, different constant domains can be fused to the V L and V H regions that contain the CDRs provided herein. In certain embodiments, the V H region that contains the CDRs provided herein can be fused to a human IgG1, IgG2, IgG3 or IgG4 heavy chain (HC) constant domain or a variant thereof that contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native or wild-type IgG1, IgG2, IgG3 or IgG4 isotype, and variants thereof in which the HC lacks the variant C-terminal lysine or glycine-lysine.

[0201] In certain embodiments, the anti-ILT3 antibody (or antigen-binding fragment) has altered effector function and can comprise a heavy chain constant domain other than the native (wild-type) human IgG1, such as a human IgG1 with mutations that abolish or minimize one or more effector functions, including the ability to bind complement, human IgG4, or a hybrid human IgG1 / human IgG4, and variants thereof in which the HC lacks the C-terminal lysine or glycine-lysine.

[0202] While native human IgG1 antibodies provide long half-lives and effector functions such as complement activation and antibody-dependent cellular cytotoxicity, such activities may not be desirable for all uses of the antibody. Thus, in certain embodiments, it is desirable that the heavy chain constant domain or Fc have little or reduced effector function ("effector-lower"). In those cases, the anti-ILT3 HC variable domain can be fused to a human IgG4 constant domain that is generally known to be effector-lower or to an IgG1 constant domain that has been mutated to be effector-lower. These effector-lower molecules have minimal or reduced binding to human FcγRIIIA, FcγRIIA, and FcγRI compared to polypeptides containing wild-type IgG Fc regions, where the affinity for human FcγRIIIA, FcγRIIA, and FcγRI is reduced 1.15 to 100-fold compared to polypeptides containing wild-type IgG constant domains, and where the antibody-dependent cell-mediated cytotoxicity (ADCC) induced by such molecules is 0-20% of the ADCC induced by polypeptides containing wild-type human IgG1 constant domains.

[0203] Accordingly, in certain embodiments, the invention includes chimeric or humanized anti-ILT3 antibodies and antigen-binding fragments thereof that comprise a human IgG4 constant domain. In another embodiment, the human IgG4 constant domain can be modified to be different at the position corresponding to position 228 in the EU system and position 241 in the Kabat system of the native (wild-type) human IgG4 constant domain (Swiss-Prot accession number P01861.1), where the native serine at position 108 (Ser108) of the HC constant domain is replaced with proline (Pro), see, e.g., SEQ ID NO:9. This modification prevents the formation of potential interchain disulfide bonds between cysteine (Cys106) at position 106 and cysteine (Cys109) at position 109, which corresponds to Cys226 and Cys229 in the EU system and Cys239 and Cys242 in the Kabat system, and which may interfere with proper intrachain disulfide bond formation. See Angal et al. Mol. Immunol. 30:105 (1993); see also (Schuurman et al., Mol. Immunol. 38:1-8, (2001); SEQ ID NO:14 and 41). In certain embodiments, the human IgG4 constant domain can further include an L235E substitution in addition to the S228P substitution.

[0204] In another embodiment, a chimeric or humanized anti-ILT3 antibody can be fused to a modified human IgG1 constant domain that has been modified to be less effector. In one embodiment, the human IgG1 HC can contain substitutions of human IgG2 HC residues at positions 233-236 and IgG4 HC residues at positions 327, 330, and 331 to greatly reduce ADCC and CDC (Armour et al., Eur J Immunol. 29(8):2613-24(1999); Shields et al., J Biol Chem. 276(9):6591-604(2001)). In certain embodiments, the antibody comprises a human IgG1 heavy chain (HC) constant domain or variant thereof that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of native IgG, which provides an antibody with reduced or minimal effector function. In certain aspects, IgG1 has been modified to contain the L234A, L235A, and D265S mutations or consists of the L234A, L235A, and D265S mutations to make the Fc effector less. Other mutations useful for making the IgG1 Fc effector less can be found in U.S. Patent No. 8,969,526.

[0205] In another embodiment, the human IgG1 HC is modified to lack an asparagine (Asn) residue near position 297 of the HC for N-glycosylation. The consensus sequence for N-glycosylation is Asn-Xaa-Ser / Thr (where Xaa is any amino acid other than Pro); in IgG1, the N-glycosylation consensus sequence is Asn-Ser-Thr. The modification can be achieved by replacing the codon for Asn at position 297 in the nucleic acid molecule encoding the HC with the codon for another amino acid such as Gln. Alternatively, the codon for Ser can be replaced with the codon for Pro or the codon for threonine can be replaced with any codon other than the codon for Ser, e.g., N297A or N297D. Such a modified IgG1 molecule has little or no detectable effector function. Alternatively, all three codons are modified.

[0206] In another embodiment, the human IgG1 constant domain is modified to contain one or more amino acid substitutions selected from E233P, L234A, L235A, L235E, N297A, N297D, D265S, and P331S, where the residues are according to the EU index numbering of Kabat, and where the polypeptide shows a reduced affinity for human FcγRIIIa and / or for FcγRIIa and / or FcγRI compared to a polypeptide comprising a wild-type IgG constant domain region. In a particular embodiment, the human IgG constant domain contains, for example, the substitutions L234A, L235A, and D265S as shown in SEQ ID NO:4. In a particular embodiment, the human IgG1 constant domain contains an amino acid substitution at position Pro329 and at least one other amino acid substitution from E233P, L234A, L235A, L235E, N297A, N297D, D265S, and P331S. These and other substitutions are disclosed in WO9428027; WO2004099249; WO20121300831, U.S. Patent Nos. 9,708,406; 8,969,526; 9,296,815; Sondermann et al. Nature 406, 267-273 (20 Jul. 2000).

[0207] In one embodiment of the invention, the anti-ILT3 antibody or antigen-binding fragment includes embodiments where one or more of the six CDRs have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, including a full tetrameric structure having two light chains and two heavy chains (including the constant regions). The variable regions of each light chain / heavy chain pair form the antibody-binding site. Thus, generally, the intact antibody has two binding sites. Except for bispecific antibodies, the two binding sites are usually identical.

[0208] In a particular embodiment, the invention provides the anti-ILT3 antibodies shown in Table 4. Except for those antibodies that contain a substitution of a tryptophan residue at position 101 of V H the antibodies disclosed herein bind human ILT3.

[0209]

[0210]

[0211]

[0212]

[0213]

[0214] As described in Example 4, epitope mapping by hydrogen-deuterium exchange mass spectrometry (HDX-MS) showed that the anti-ILT3 antibodies disclosed herein bind to an epitope on the extracellular domain of ILT3 near the boundary between the D1 and D2 domains. The epitope determined using HDX-MS indicates that the epitope bound by the anti-ILT3 antibodies disclosed herein comprises or consists of at least one amino acid within one or more of the peptide domain amino acid sequences selected from SEQ ID NOs: 3, 4, 5, 6, 7, and 8. In a further embodiment, the epitope comprises or consists of one or more of the peptide domain amino acid sequences selected from SEQ ID NOs: 3, 4, 5, 6, 7, and 8. In certain embodiments, the epitope comprises or consists of each of the peptide domain amino acid sequences selected from SEQ ID NOs: 3, 4, 5, 6, 7, and 8 and determined in HDX-MS. In a specific embodiment, the epitope comprises or consists of one or more of the peptide domain amino acid sequences selected from SEQ ID NOs: 3, 4, 5, 6, 7, and 8. In a particular embodiment, the epitope comprises or consists of the peptide domains shown in SEQ ID NOs: 3, 4, 5, 6, 7, and 8.

[0215] The invention also provides chimeric, humanized or human antibodies or antigen-binding fragments thereof that bind to an epitope on ILT3, wherein the epitope comprises or consists of at least one amino acid within one or more of the peptide domains comprising the amino acid sequences shown in SEQ ID NOs: 3, 4, 5, 6, 7, and 8 as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis.

[0216] In another embodiment, the invention further provides chimeric, humanized or human antibodies or antigen-binding fragments thereof that bind to an epitope on ILT3, wherein the epitope comprises or consists of amino acids within one or more of the peptide domains shown in SEQ ID NOs: 3, 4, 5, 6, 7, and 8. In certain embodiments, the epitope comprises or consists of each of the peptide domains determined and shown in the Figure 3A heat map determined by HDX-MS.

[0217] The present invention further provides chimeric, humanized or human antibodies or antigen-binding fragments thereof that cross-block antibodies binding to an epitope on ILT3, said antibodies comprising a heavy chain variable domain having the amino acid sequence shown in SEQ ID NO:15 and a light chain variable domain having the amino acid sequence shown in SEQ ID NO:16. In a further embodiment, the epitope comprises or consists of at least one amino acid within one or more of the peptide domains, said peptide domains comprising or consisting of the amino acid sequences shown in SEQ ID NO:3, 4, 5, 6, 7, and 8 as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis. In a further embodiment, the epitope comprises or consists of amino acids within one or more of the peptide domains shown in SEQ ID NO:3, 4, 5, 6, 7, and 8. In certain embodiments, the epitope comprises or consists of at least one amino acid within each of the peptide domains as determined by HDX-MS.

[0218] The present invention further provides bispecific antibodies and antigen-binding fragments thereof that comprise a first antibody or antigen-binding fragment that binds ILT3 and a second antibody or antigen-binding fragment that binds a molecule other than ILT3, wherein the first antibody or antigen-binding fragment comprises at least an HC-CDR3 having an amino acid sequence selected from SEQ ID NO:22, 49, 57, 65, 73, 81, 89, 97, and 105, or an amino acid sequence having 3, 2, or 1 amino acid differences from the amino acid sequences selected from SEQ ID NO:22, 49, 57, 65, 73, 81, 89, 97, and 105, wherein the first antibody binds an ILT3 epitope that comprises amino acids within the sequences of SEQ ID NO:3, 4, 5, 6, 7, and 8, and the second antibody binds a molecule other than ILT3, and methods of using the same.

[0219] The present invention further provides bispecific antibodies and antigen-binding fragments thereof that comprise a first antibody or antigen-binding fragment that binds ILT3 and a second antibody or antigen-binding fragment that binds a molecule other than ILT3, wherein the first antibody or antigen-binding fragment comprises at least six CDRs of an anti-ILT3 antibody or an embodiment thereof wherein one or more of the CDRs have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, and wherein the first antibody binds an ILT3 epitope that comprises amino acids within the sequences of SEQ ID NO:3, 4, 5, 6, 7, and 8, and the second antibody binds a molecule other than ILT3, and methods of using the same.

[0220] The present invention further provides a bispecific antibody (an antibody having binding specificity for different epitopes on the same antigen), which has a first heavy chain / light chain pair of a first antibody, which comprises at least HC-CDR3, and the HC-CDR3 has an amino acid sequence selected from SEQ ID NO: 22, 49, 57, 65, 73, 81, 89, 97, and 105, or has an amino acid sequence having 3, 2, or 1 difference from the amino acid sequences selected from SEQ ID NO: 22, 49, 57, 65, 73, 81, 89, 97, and 105; wherein the first heavy chain / light chain pair binds to an ILT3 epitope, the epitope comprising amino acids within the sequences of SEQ ID NO: 3, 4, 5, 6, 7, and 8, and the second antibody binds to a molecule other than ILT3, and the second heavy chain / light chain pair of the second antibody has specificity for an anti-ILT3 epitope different from the epitope recognized by the first heavy / light chain pair.

[0221] The present invention further provides a bispecific antibody (an antibody having binding specificity for different epitopes on the same antigen), which has a first heavy chain / light chain pair of a first antibody, which comprises at least six CDRs of an anti-ILT3 antibody or an embodiment thereof in which one or more of the CDRs have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, wherein the first antibody binds to an ILT3 epitope, the epitope comprising amino acids within the sequences of SEQ ID NO: 3, 4, 5, 6, 7, and 8, wherein the first heavy chain / light chain pair binds to an ILT3 epitope, the epitope comprising amino acids within the sequences of SEQ ID NO: 3, 4, 5, 6, 7, and 8, and the second antibody binds to a molecule other than ILT3, and the second heavy chain / light chain pair of the second antibody has specificity for an anti-ILT3 epitope different from the epitope recognized by the first heavy / light chain pair.

[0222] Drug compositions and administration

[0223] To prepare a pharmaceutical or sterile composition of an anti-ILT3 antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment is mixed with a pharmaceutically acceptable carrier or excipient. See, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984) and U.S. Pharmacopeial Convention (USP) 12601 Twinbrook Parkway, Rockville, MD 20852-1790, USA, which is updated continuously online.

[0224] Preparations of therapeutic and diagnostic agents can be prepared by mixing them with a pharmaceutical carrier, excipient, or stabilizer in the following forms, for example, lyophilized powders, slurries, aqueous solutions, or suspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman’s The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

[0225] In another embodiment, a composition comprising an antibody or antibody fragment disclosed herein is administered to a subject according to Physicians' Desk Reference 2017 (Thomson Healthcare; 75th Edition (November 1, 2002)). Methods of administering antibody molecules are known in the art and are described below. The appropriate dosage of the molecule used will depend on the age and weight of the subject and the particular drug used. The dosage and treatment regimen of the anti-ILT3 antibody or antigen-binding fragment can be determined by one skilled in the art. In certain embodiments, the anti-ILT3 antibody or antigen-binding fragment is administered by injection (e.g., subcutaneously or intravenously) at a dosage of about 1 to 30 mg / kg, such as about 5 to 25 mg / kg, about 10 to 20 mg / kg, about 1 to 5 mg / kg or about 3 mg / kg. In some embodiments, the anti-ILT3 antibody or antigen-binding fragment is at about 1 mg / kg, about 3 mg / kg or 10 mg / kg, about 20 mg / kg, about 30 mg / kg or about 40 mg / kg. In some embodiments, the anti-ILT3 antibody or antigen-binding fragment is administered at a dosage of about 1-3 mg / kg or about 3-10 mg / kg. In some embodiments, the anti-ILT3 antibody or antigen-binding fragment is administered at a dosage of about 0.5-2, 2-4, 2-5, 5-15 or 5-20 mg / kg. The dosing schedule can vary from, for example, once a week to once every 2, 3 or 4 weeks. In one embodiment, the anti-ILT3 antibody or antigen-binding fragment is administered at a dosage of about 10 to 20 mg / kg every other week.

[0226] The mode of administration can vary. Suitable routes of administration are preferably parenteral or subcutaneous. Other routes of administration can include oral, transmucosal, intradermal, direct intraventricular, intravenous, intranasal, inhalation, insufflation or intra-arterial.

[0227] In certain embodiments, the anti-ILT3 antibody or its antigen-binding fragment can be administered by an invasive route such as by injection. In other embodiments of the invention, the anti-ILT3 antibody or its antigen-binding fragment or its pharmaceutical composition can be administered intravenously, subcutaneously, intra-arterially or by inhalation, aerosol delivery. Administration by non-invasive routes (e.g., oral; e.g., in pills, capsules or tablets) is also within the scope of the present invention.

[0228] The composition can be administered with a medical device known in the art. For example, the pharmaceutical composition of the present invention can be administered by injection with a subcutaneous injection needle, which includes, for example, a pre-filled syringe or an autoinjector.

[0229] The pharmaceutical compositions disclosed herein may also be administered with a needleless subcutaneous injection device; preferably, a needleless subcutaneous injection device may be used. For example, the devices disclosed in U.S. Patent Nos. 6,620,135; 6,096,002; 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.

[0230] The pharmaceutical compositions disclosed herein may also be administered by infusion. Examples of well-known implants and modules for administering pharmaceutical compositions include: U.S. Patent No. 4,487,603 discloses an implantable microinfusion pump for dispensing a drug at a controlled rate; U.S. Patent No. 4,447,233 discloses a drug infusion pump for delivering a drug at a precise infusion rate. U.S. Patent No. 4,447,224 discloses a variable flow implantable infusion device for continuous drug delivery. U.S. Patent. U.S. Patent 4,439,196 discloses an osmotic drug delivery system having multiple chambers. Many other such implants, delivery systems and modules are well known to those skilled in the art.

[0231] The dosing regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic antibody, the level of symptoms, the immunogenicity of the therapeutic antibody, and the accessibility of target cells in the biological matrix. Preferably, the dosing regimen delivers sufficient therapeutic antibody to effect an improvement in the target disease state while minimizing undesirable side effects. Thus, the amount of biologic agent delivered depends in part on the particular therapeutic antibody and the severity of the condition being treated. There are guidelines for selecting the appropriate dose of a therapeutic antibody (see, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert, et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343:1594-1602).

[0232] Adjust the dosage regimen to provide the optimal desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several separate doses may be administered over time, or the dosage may be proportionally decreased or increased depending on the urgency of the treatment situation. Formulating the parenteral composition in dosage unit form is particularly advantageous for ease of administration and uniform dosage. As used herein, dosage unit form refers to physically discrete units suitable as unit doses for the subject to be treated; e.g., a unit dose. Each unit contains a predetermined quantity of the active compound which is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification of the dosage unit forms described herein is directly determined by (a) the unique characteristics of the antibody or antibody-binding fragment and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of formulation. The sensitivity of such active molecules for treating an individual. (See, e.g., Yang, et al. (2003) New Engl. J. Med. 349:427-434; Herold, et al. (2002) New Engl. J. Med. 346:1692-1698; Liu, et al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456; Portielji, et al. (20003) Cancer Immunol. Immunother. 52:133-144).

[0233] Use of the anti-ILT3 antibody or antigen-binding fragment disclosed herein

[0234] The anti-ILT3 antibodies and antigen-binding fragments disclosed herein that are not admixed with related ILTs can be used in conventional immunoassays such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or tissue immunohistochemistry for the specific detection of human ILT3 (e.g., in a biological sample such as serum or plasma). The invention thus provides a method for detecting human ILT3, comprising contacting a biological sample with an anti-ILT3 antibody or antigen-binding fragment in the biological sample and detecting either the anti-ILT3 antibody or antigen-binding fragment bound to human ILT3 or the unbound anti-ILT3 antibody or antigen-binding fragment disclosed herein, thereby detecting human ILT3 in the biological sample. The anti-ILT3 antibody or antigen-binding fragment is directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound anti-ILT3 antibody or antigen-binding fragment disclosed herein. Suitable detectable substances include a variety of enzymes, cofactors, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; and the like. Examples of suitable bridging complex groups include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. Examples of luminescent materials include luminol; examples of suitable radioactive materials include 125I, 131I, 35S, and 3H.

[0235] In addition to labeling the anti-ILT3 antibody or antigen-binding fragment, human ILT3 can be assayed in biological fluids by a competitive immunoassay using an ILT3 standard labeled with a detectable substance and an unlabeled anti-human ILT3 anti-ILT3 antibody or disclosed antigen-binding fragment. In this assay, a biological sample, the labeled ILT3 standard, and the anti-ILT3 antibody or antigen-binding fragment are combined, and the amount of labeled ILT3 standard bound to the unlabeled anti-ILT3 antibody or antigen-binding fragment disclosed herein is determined. The amount of human ILT3 in the biological sample is inversely proportional to the amount of anti-ILT3 antibody or antigen-binding fragment bound to the labeled standard ILT3.

[0236] The anti-ILT3 antibodies or antigen-binding fragments disclosed herein can also be used to detect ILT3 from species other than human, particularly ILT3 from primates (e.g., cynomolgus monkey or rhesus monkey).

[0237] Methods for upregulating the immune response in vivo

[0238] The anti-ILT3 antibodies or antigen-binding fragments disclosed herein can be used as immunostimulatory compositions, e.g., used alone or as part of a vaccine or in combination therapy to promote the activation of B cells and / or T cells, e.g., either Th1 cell or Th2 cell activation. That is, the anti-ILT3 antibodies or antigen-binding fragments disclosed herein can be used as adjuvants for use in combination with an antigen of interest to enhance the immune response against that antigen in vivo. For example, to stimulate an antibody or cellular immune response to an antigen of interest (e.g., for vaccination purposes), the anti-ILT3 antibodies or antigen-binding fragments disclosed herein can be co-administered (e.g., co-administered simultaneously in the same or different compositions, or sequentially in time to enhance the immune response). The antigen of interest and the anti-ILT3 antibodies or antigen-binding fragments disclosed herein can be formulated together as a single pharmaceutical composition or as separate compositions. In one embodiment, the antigen of interest and the anti-ILT3 antibodies or antigen-binding fragments disclosed herein are administered to a subject simultaneously. Alternatively, in some cases, it may be necessary to administer the antigen first and then the anti-ILT3 antibodies or antigen-binding fragments disclosed herein, or vice versa (e.g., in the case of an antigen that naturally elicits a Th1 response, it may be advantageous to first administer the antigen alone to stimulate the Th1 response and then administer the anti-ILT3 antibodies or antigen-binding fragments disclosed herein alone or in combination with the antigen boost to shift the immune response to a Th2 response). In a preferred embodiment, the anti-ILT3 antibodies or antigen-binding fragments disclosed herein are administered upon priming with the antigen, i.e., at the time of the first antigen administration. For example, days -3, -2, -1, 0, +1, +2, +3. A particularly preferred day of administration of the anti-ILT3 antibodies or antigen-binding fragments disclosed herein is day -1.

[0239] In one embodiment, the anti-ILT3 antibodies or antigen-binding fragments disclosed herein are administered with an antigen of interest. The antigen of interest is an antigen against which an immune response is desired. For example, an antigen of interest is an antigen that can stimulate immune protection against an infectious agent derived from that antigen in a subject challenged with the agent. It is also contemplated to administer the anti-ILT3 antibodies or antigen-binding fragments disclosed herein to increase the immune response without administering an antigen.

[0240] Thus, exemplary antigens of interest include those derived from infectious agents, where the immune response against the antigen is used to prevent or treat a disease caused by that agent. Such antigens include, but are not limited to, viral, bacterial, fungal, or parasitic proteins, as well as any other proteins, glycoproteins, lipoproteins, glycolipids, etc. Antigens of interest also include those that provide a benefit to a subject at risk of acquiring or diagnosed with a tumor. The subject is preferably a mammal, most preferably a human.

[0241] Typical antigens of interest can be classified as follows: protein antigens such as ceruloplasmin and serum albumin; and bacterial antigens such as cholanic acid, flagellar antigens, capsular polysaccharides, extracellular bacterial products and toxins; glycoproteins and glycolipids; viruses such as animal, plant and bacterial viruses; bound and synthetic antigens such as protein / hapten conjugates, which are molecules preferentially expressed by tumors as compared to normal tissues; synthetic polypeptides; nucleic acids such as ribonucleic acid and deoxyribonucleic acid. As used herein, the term "infectious agent" includes any agent that expresses an antigen and elicits a cellular immune response in a host. Non-limiting examples of viral antigens that may be considered useful include, but are not limited to, the nucleoprotein (NP) of influenza virus and the Gag protein of HIV. Other heterologous antigens include, but are not limited to, the HIV Env protein or its components gp120 and gp41, the HIV Nef protein and the HIV Pol protein, reverse transcriptase and protease. In addition, other viral antigens such as Ebola virus (EBOV) antigens such as EBOV NP or glycoprotein (GP), full-length or GP-deleted in the mucin region of the molecule (Yang et al., Nat MED 6:886 (2000)), smallpox antigens, hepatitis A, B or C virus, human rhinoviruses such as type 2 or 14, herpes simplex virus, poliovirus type 2 or 3, foot-and-mouth disease virus (FMDV), rabies virus, rotavirus, influenza virus, coxsackievirus, human papillomavirus (HPV) (e.g., type 16 papillomavirus), its E7 protein and fragments containing the E7 protein or its epitopes; and simian immunodeficiency virus (SIV) can be used. Antigens of interest need not be limited to antigens of viral origin. Parasite antigens such as, for example, malaria antigens are included, as are fungal antigens, bacterial antigens and tumor antigens. Examples of antigens derived from bacterial antigens are those from Bordetella pertussis (e.g., P69 protein and filamentous hemagglutinin (FHA) antigens), Vibrio cholerae, Bacillus anthracis, and antigens of Escherichia coli such as Escherichia coli (E. coli) heat-labile toxin B subunit (LT-B), E. coli K88 antigen, and enterotoxigenic Escherichia coli (E. coli) antigens. Other examples of antigens include Schistosoma mansoni P28 glutathione S-transferase antigen (P28 antigen) and antigens of trematodes, mycoplasmas, Ascaris, tapeworms, Chlamydia trachomatis and malaria parasites such as, for example, the genus Plasmodium or Babesia such as Plasmodium falciparum, and peptides encoding immunogenic epitopes of the above antigens.

[0242] As used herein, the term "tumor-associated antigen" refers to an antigen that affects tumor growth or metastasis in a host organism. A tumor-associated antigen can be an antigen expressed by a tumor cell, or can be an antigen expressed by a non-tumor cell, but when so expressed, can promote the growth or metastasis of tumor cells. The types of tumor antigens and tumor-associated antigens include any known or heretofore unknown tumor antigens, including but not limited to the bcr / abl antigen in leukemia, the HPVE6 and E7 antigens of oncogenic viruses associated with cervical cancer, the MAGE1 and MZ2-E antigens in or associated with melanoma, and the MVC-1 and HER-2 antigens in or associated with breast cancer.

[0243] Infections, diseases or disorders that can be treated or prevented by administering a composition comprising an anti-ILT3 antibody or antigen-binding fragment disclosed herein include any infection, disease or disorder in which the host immune response plays a role in preventing the infection, disease or disorder. Disorders. Diseases, disorders or infections that can be treated or prevented by administering a composition comprising an anti-ILT3 antibody or antigen-binding fragment disclosed herein include, but are not limited to, any infection, disease or disorder caused by or associated with bioterrorism, listeriosis, Ebola virus, SARS, smallpox, hepatitis A, hepatitis B, hepatitis C, human-induced diseases and disorders, fungi, parasites, viruses or bacteria, diseases, disorders or infections caused by or associated with rotavirus, influenza, coxsackievirus, human papillomavirus, SIV, malaria, cancer (e.g., tumors), rhinovirus, HIV and AIDS, herpes, polio, foot-and-mouth disease, rabies, diseases or disorders caused by Bordetella pertussis, Vibrio cholerae, Bacillus anthracis, Escherichia coli, trematodes, mycoplasmas, vibrios, tapeworms, Chlamydia trachomatis and Plasmodium falciparum, etc.

[0244] Immune response tumor cells

[0245] Regulatory T cells play an important role in maintaining immunological self-tolerance by suppressing immune responses against autoimmune diseases and cancer. Thus, in one embodiment, upregulating the immune response would be beneficial for enhancing the immune response in cancer. Accordingly, the anti-ILT3 antibody or antigen-binding fragment disclosed herein can be used in the treatment of malignancies to inhibit tumor growth or metastasis. The anti-ILT3 antibody or antigen-binding fragment disclosed herein can be administered systemically or locally to the tumor site.

[0246] In one embodiment, modulation of human ILT3 function can be used to induce tumor immunity. An ILT3-binding molecule can be administered to a patient having tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) to overcome tumor-specific tolerance in the subject.

[0247] As used herein, the term "neoplastic disease" is characterized by malignant tumor growth or a disease state characterized by benign hyperproliferation and proliferative cells. The general medical meaning of the term "neoplasia" refers to "new cell growth" that results in a loss of responsiveness to normal growth control (e.g., tumor cell growth).

[0248] As used herein, the terms "hyperproliferation" and "proliferative" are used interchangeably and refer to an abnormal state or condition in those cells characterized by rapid proliferation or tumors. The terms "malignant" and "neoplastic" are meant to include all types of hyperproliferative growth, proliferative growth, cancerous growth or carcinogenic processes, metastatic tissue or malignantly transformed cells, tissues or organs, regardless of histopathologic type or invasiveness. "Proliferation" in the context of a stage refers to cells that undergo an abnormally high rate of growth. However, as used herein, the terms "neoplasia" and "proliferation" may be used interchangeably and, as revealed by their context, generally refer to cells that undergo an abnormal rate of cell growth. Neoplasia and proliferation include "tumors", which may be benign, pre-malignant or malignant.

[0249] The terms "neoplasia", "proliferation" and "tumor" are commonly referred to as "cancer", which is a general term for over 100 diseases characterized by uncontrolled, abnormal cell growth. Examples of cancers include, but are not limited to: breast cancer; colon; non-small cell lung, head and neck; colorectal lung; prostate; ovary; renal melanoma gastrointestinal (e.g., pancreas and stomach) cancer; and osteosarcoma.

[0250] Pancreatic cancer, melanoma, breast cancer, lung cancer,: In one embodiment, the cancer is selected from head and neck cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, urothelial bladder cancer, brain or central nervous system cancer (e.g., glioblastoma), peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, renal cancer, testicular cancer, biliary tract cancer, small intestine or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma and blood tissue cancer.

[0251] Immune response to an infectious agent

[0252] Upregulation of an immune response can be in the form of enhancing an existing immune response or initiating an initial immune response. For example, in the case of a viral infection, enhancing the immune response by modulating ILT3 may be useful. Since the anti-ILT3 antibodies or antigen-binding fragments disclosed herein can act to enhance the immune response, they will be therapeutically useful in situations where more rapid or complete clearance of pathogens such as bacteria and viruses would be beneficial.

[0253] As used herein, the term "viral infection" includes the infection of the following organisms, which include but are not limited to HIV (e.g., HIV-1 and HIV-2), human herpesviruses, cytomegalovirus (especially human), rotavirus, Epstein-Barr virus, varicella-zoster virus, hepatitis B virus, such as hepatitis B virus, hepatitis A virus, hepatitis C virus, and hepatitis E virus, paramyxoviruses: respiratory syncytial virus, parainfluenza virus, measles virus, mumps virus, human papillomavirus (e.g., HPV6, 11, 16, 18, etc.), flaviviruses (e.g., yellow fever virus, dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus), or influenza virus.

[0254] As used herein, the term "bacterial infection" includes infections by various bacterial organisms, including Gram-positive and Gram-negative bacteria. Examples include, but are not limited to, Neisseria species including Neisseria gonorrhoeae and Neisseria meningitidis; Streptococcus species including Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus mutans; Haemophilus species including Haemophilus influenzae type B, Haemophilus influenzae non-typeable, Haemophilus ducreyi; Moraxella species including Moraxella catarrhalis, also known as Branhamella catarrhalis; Bordetella species including Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica; Mycobacterium species including Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium avium, Mycobacterium paratuberculosis, Mycobacterium smegmatis; Legionella species including Legionella pneumophila; Escherichia coli including enterotoxigenic E. coli, enterohemorrhagic E. coli, enteropathogenic E. coli; Vibrio cholerae including Vibrio cholerae, Shigella species including S. sonnei, Shigella dysenteriae, Shigella flexneri; Yersinia species including Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Campylobacter jejuni including Campylobacter jejuni and Escherichia coli. Salmonella species including Salmonella typhi, Salmonella paratyphi, Salmonella choleraesuis, Salmonella enteritidis; Listeria species including Listeria monocytogenes; Helicobacter pylori including Helicobacter pylori; Pseudomonas species including Pseudomonas aeruginosa, Staphylococcus species including Staphylococcus aureus, Staphylococcus epidermidis; Enterococcus species including Enterococcus faecalis, Enterococcus faecium; Clostridium species including Clostridium tetani, Clostridium botulinum, Clostridium difficile; Bacillus species including Bacillus anthracis; Corynebacterium species including Corynebacterium diphtheriae; Borrelia spp. including B. burgdorferi, B. garinii, B. afzelii, B. andersonii, B. hermsii; Ehrlichia species including E. equi and the agent of human granulocytic ehrlichiosis; Rickettsia species including R. rickettsii; Chlamydia species including Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci; Leptospira spp., including L. interrogans; Treponema species including Treponema pallidum, Treponema denticola, Treponema hyodysenteriae. Preferred bacteria include, but are not limited to, Listeria, Mycobacterium, Mycobacterium (e.g., tuberculosis), anthrax, Salmonella, and Listeria monocytogenes.

[0255] In another embodiment, T cells can be removed from a patient and contacted in vitro with an anti-ILT3 antibody or antigen-binding fragment disclosed herein, optionally with an activating signal (e.g., antigen plus APC or polyclonal antibody) and reintroduced into the patient.

[0256] The anti-ILT3 antibodies or antigen-binding fragments disclosed herein can also be prophylactically used in vaccines against various pathogens. Immunity against pathogens such as viruses can be induced by vaccination with viral proteins and the anti-ILT3 antibodies or antigen-binding fragments disclosed herein. Alternatively, expression vectors, i.e., genes encoding the antigens and anti-ILT3 antibodies or antigen-binding fragments disclosed herein, for example, vaccinia virus expression vectors engineered to express viral proteins encoding nucleic acids and anti-ILT3 antibodies or antigen-binding fragments encoding nucleic acids disclosed herein, can be used for vaccination. Pathogens for which the vaccines may be useful include, for example, hepatitis B, hepatitis C, Epstein-Barr virus, cytomegalovirus, HIV-1, HIV-2, tuberculosis, malaria, and schistosomiasis.

[0257] The invention further includes anti-ILT3 antibodies or antigen-binding fragments disclosed herein conjugated to a diagnostic or therapeutic agent. The anti-ILT3 antibodies or antigen-binding fragments disclosed herein can be used for diagnosis as part of a clinical test procedure, for example, to monitor the occurrence or progression of a tumor, to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by conjugating the antibody to a detectable substance. Examples of detectable substances include various enzymes, cofactors, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals using various positron emission tomography, and non-radioactive paramagnetic metal ions. The detectable substances can be directly or indirectly conjugated or bound to the binding molecule using techniques known in the art through an intermediate (such as a linker known in the art). U.S. Patent No. 4,741,900 discloses metal ions that can be bound to a binding molecule. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; and so on. Examples of suitable repair group complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. Examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; examples of suitable radioactive materials are 125 I, 131 I, and 99 Tc.

[0258] In addition, the anti-ILT3 antibodies or antigen-binding fragments disclosed herein can be conjugated to a therapeutic moiety, such as a cytotoxin, such as a cell growth inhibitor or cytocide, a therapeutic agent, or a radio-metallic ion, such as an α-emitter, such as, for example, 213Bi. Cytotoxins or cytotoxic agents include any substance that is detrimental to cells. Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dactinomycin, mitoxantrone, mithramycin, procarbazine, mechlorethamine, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarboxylate), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., actinomycin D), bleomycin, mithramycin and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

[0259] The invention further relates to a method of treating, detecting, and / or preventing one or more of the following in an animal, preferably a mammal, and most preferably a human patient, by administering an anti-ILT3 antibody or antigen-binding fragment disclosed herein: the diseases, disorders, or conditions disclosed herein. The therapeutic compounds of the invention include, but are not limited to, the anti-ILT3 antibodies or antigen-binding fragments disclosed herein. The anti-ILT3 antibodies or antigen-binding fragments disclosed herein can be used to treat, diagnose, inhibit, or prevent diseases, disorders, or conditions associated with abnormal activity of ILT3, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein.

[0260] The anti-ILT3 antibodies or antigen-binding fragments disclosed herein can be advantageously used in combination with other monoclonal or chimeric binding molecules, or with lymphokines or hematopoietic growth factors (e.g., IL-2, IL-3, and IL-7). For example, it is used to increase the number or activity of effector cells that interact with the binding molecule.

[0261] The anti-ILT3 antibodies or antigen-binding fragments disclosed herein can be administered alone or in combination with other types of treatment, such as immunostimulatory therapy or therapy designed to control the proliferation of targets of activated immune cells (e.g., cancer cells or pathogens). Exemplary therapies include, for example, radiotherapy, chemotherapy, hormone therapy, immunotherapy, and anti-tumor agents, antibiotics, and immunoglobulins.

[0262] For therapeutic purposes, the n anti-ILT3 antibodies or antigen-binding fragments disclosed herein can be administered to human subjects. Moreover, the anti-ILT3 antibodies or antigen-binding fragments disclosed herein can be administered to non-human mammals that express ILT3, for veterinary purposes or as animal models of human diseases, in which the binding molecule cross-reacts with ILT3 (e.g., primates).

[0263] Combination regimens

[0264] The anti-ILT3 antibodies or antigen-binding fragments herein can be used in an uncoupled form or conjugated to a second reagent, such as a cytotoxic drug, a radioisotope, or a protein such as a protein toxin or a viral protein. The method includes: administering the anti-ILT3 antibodies or antigen-binding fragments herein, either alone or conjugated to a cytotoxic drug, to a subject in need of such treatment. The anti-ILT3 antibodies or antigen-binding fragments herein can be used to deliver a variety of therapeutic agents, such as cytotoxic moieties, such as therapeutic drugs, radioisotopes, molecules or biological proteins (such as protein toxins) or particles (such as recombinant viral particles, e.g., via viral coat proteins) of plant, fungal, or bacterial origin, or mixtures thereof.

[0265] Other combination therapies

[0266] The anti-ILT3 antibodies or antigen-binding fragments disclosed herein can be used in combination with other therapies. For example, combination therapies can include compositions comprising an anti-ILT3 antibody or antigen-binding fragment co-formulated and / or co-administered with one or more other therapeutic agents, such as one or more anti-cancer agents, cytotoxic or cytostatic agents, hormonal therapies, vaccines, and / or other immunotherapies. In other embodiments, the anti-ILT3 antibody or its antigen-binding fragment is administered with other therapeutic modalities, including surgery, radiation, cryosurgery, and / or hyperthermia. Such combination therapies can advantageously utilize lower doses of the administered therapeutic agents, thereby avoiding possible toxicities or complications associated with the various single therapies.

[0267] "In combination with" does not imply that the therapies and / or therapeutic agents must be administered simultaneously and / or formulated for co-delivery, although such delivery methods are within the scope described herein. The anti-ILT3 antibody or its antigen-binding fragment can be administered either before or after the administration of one or more other additional therapies or therapeutic agents. The anti-ILT3 antibody or its antigen-binding fragment and the other agents or treatment regimens can be administered in any order. Generally, each agent will be administered at the dose and / or schedule determined for that agent. It will also be recognized that the additional therapeutic agents used in the combination can be administered together in a single composition or separately in different compositions. Generally, it is desirable to use the additional therapeutic agents in combination at levels not exceeding those at which they are used alone. In some embodiments, the levels used in combination will be lower than those used alone.

[0268] In certain embodiments, the anti-ILT3 antibodies or antigen-binding fragments thereof described herein are administered in combination with one or more checkpoint inhibitors or antagonists of programmed death receptor 1 (PD-1) or its ligands PD-L1 and PD-L2. The inhibitor or antagonist can be an antibody, antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide. In some embodiments, the anti-PD-1 antibody is selected from nivolumab (OPDIVO, Bristol Myers Squibb, New York, NY), pembrolizumab (KEYTRUDA, Merck Sharp & Dohme Corp, Kenilworth, NJ, USA), cemiplimab (Regeneron, Tarrytown, NY), or pidilizumab (CT-011). In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising the extracellular or PD-1-binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 inhibitor is AMP-224. In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody, such as durvalumab (IMFINZI, Astrazeneca, Wilmington, DE), atezolizumab (TECENTRIQ, Roche, Zurich, CH), or avelumab (BAVENCIO, EMD Serono, Billerica, MA). In some embodiments, the anti-PD-L1 binding antagonist is selected from YW243.55.S70, MPDL3280A, MEDI-4736, MSB-0010718C, or MDX-1105.

[0269] MDX-1105, also known as BMS-936559, is the anti-PD-L1 antibody described in WO2007 / 005874. The antibody YW243.55.S70 is an anti-PD-L1 (the heavy and light chain variable region sequences are shown as SEQ ID NO:20 and 21, respectively) described in WO2010 / 077634.

[0270] Nivolumab, also known as OPDIVO, MDX-1106-04, ONO-4538, or BMS-936558, is a fully human IgG4 anti-PD-1 antibody described in WO2006 / 121168 and U.S. Patent 8,008,449.

[0271] Pembrolizumab, also known as KEYTRUDA, Ambrolizumab, MK-3475 or SCH-900475, is a humanized anti-PD-1 antibody, described in U.S. Patent No. 8,354,509 and WO2009 / 114335, and disclosed, for example, in Hamid, et al., New England J. Med. 369(2):134-144(2013). The heavy and light chains of pembrolizumab are represented by the amino acid sequences shown in SEQ ID Nos: 225 and 226, respectively.

[0272] Pidilizumab, also known as CT-011 (CureTech), is a humanized IgG1 monoclonal antibody that binds to PD-1. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in WO2009 / 101611. Other anti-PD-1 antibodies include AMP514 (Amplimmune), especially the anti-PD-1 antibodies disclosed in U.S. Patent 8609089; U.S. Publication No. 2010028330; and U.S. Publication No. 20120114649.

[0273] AMP-224 (B7-DCIg; Amplimmune; disclosed, for example, in WO2010 / 027827 and WO2011 / 066342) is a PD-L2Fc fusion soluble receptor that blocks the interaction between PD-1 and B7-H1.

[0274] MDPL3280A (Genentech / Roche) is a human Fc-optimized IgG1 monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies against PD-L1 are disclosed in U.S. Patent No. 7,943,743 and U.S. Publication No. 20120039906.

[0275] Other anti-PD-L1 binding agents include YW243.55.S70 (the heavy and light chain variable regions are shown as SEQ ID NO: 20 and 21 in WO2010 / 077634) and MDX-1105 (also known as BMS-936559). It and other anti-PD-L1 binding agents are disclosed in WO2007 / 005874.

[0276] Kit

[0277] Also provided are kits comprising one or more components, said components including but not limited to the anti-ILT3 antibodies or antigen-binding fragments thereof described herein, and one or more other components, including but not limited to another therapeutic agent. As described herein. The antibody or fragment and / or therapeutic agent can be formulated in a pharmaceutical composition as a pure composition or in combination with a pharmaceutically acceptable carrier.

[0278] In one embodiment, the kit comprises the anti-ILT3 antibody or antigen-binding fragment thereof or a pharmaceutical composition thereof in one container (e.g., in a sterile glass or plastic vial), and another therapeutic agent in another container (e.g., in a sterile glass or plastic vial).

[0279] In another embodiment, the kit comprises the combination anti-ILT3 antibody or antigen-binding fragment thereof or a pharmaceutical composition thereof formulated together, optionally, in a pharmaceutical composition, in a single, common container.

[0280] If the kit comprises a pharmaceutical composition for parenteral administration to a subject, the kit can include a device for effecting such administration. For example, the kit can include one or more subcutaneous injection needles or other injection devices as described above. Accordingly, the invention includes kits that include an injection device and an anti-ILT3 antibody or antigen-binding fragment thereof, e.g., wherein the injection device comprises the antibody or fragment, or wherein the antibody or fragment is in a separate container.

[0281] The kit can include a package insert that includes information about the pharmaceutical composition and dosage form in the kit. Generally, such information aids patients and physicians in the effective and safe use of the enclosed pharmaceutical composition and dosage form. For example, the following information about the combination of the present invention can be provided in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdose, appropriate dosage and mode of administration, appropriate storage conditions provided, references, manufacturer / distributor information and patent information.

[0282] Methods for preparing antibodies and antigen-binding fragments thereof

[0283] The anti-ILT3 antibodies or antigen-binding fragments thereof disclosed herein can also be produced recombinantly. In this embodiment, a nucleic acid molecule encoding the antibody molecule can be inserted into a vector (plasmid or virus) and transfected or transformed into a host cell, in which the nucleic acid molecule can be expressed and secreted from the host cell. Several methods for producing recombinant antibodies are known in the art.

[0284] In certain aspects, the present invention provides nucleic acid molecules encoding HC and LC, wherein the HC comprises at least HC-CDR3 of the anti-ILT3 antibody disclosed herein or an embodiment thereof wherein the HC-CDR3 has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof. In further embodiments, the HC and / or LC variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

[0285] In certain aspects, the present invention provides nucleic acid molecules encoding HC and LC, wherein the HC comprises HC-CDR1, 2, and 3 of the anti-ILT3 antibody disclosed herein or an embodiment thereof wherein one or more of HC-CDR1, 2, and 3 has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, and wherein the LC comprises LC-CDR1, 2, and 3 of the anti-ILT3 antibody disclosed herein or an embodiment thereof wherein one or more of HC-CDR1, 2, and 3 has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof. In further embodiments, the HC and / or LC variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

[0286] In certain aspects, the present invention provides a first expression vector comprising a nucleic acid molecule encoding HC, the HC comprising at least the HC CDR of the anti-ILT3 antibody disclosed herein or an embodiment thereof wherein one or more of the three HC CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, and / or wherein the HC variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof, and a second expression vector comprising a nucleic acid molecule encoding LC, the LC comprising at least the LC CDR of the anti-ILT3 antibody disclosed herein or an embodiment thereof wherein one or more of the three LC CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, and / or wherein the LC variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

[0287] In certain aspects, the present invention provides nucleic acid molecules encoding V H and V L wherein V H comprises at least HC-CDR3 of the anti-ILT3 antibody disclosed herein or an embodiment thereof wherein the HC-CDR3 has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof. In further embodiments, V H and / or V LThe variable region framework contains 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

[0288] In certain aspects, the present invention provides nucleic acid molecules encoding for V H and V L wherein the HC comprises HC-CDR1, 2, and 3 of the anti-ILT3 antibody disclosed herein or an embodiment wherein one or more of HC-CDR1, 2, and 3 has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, and wherein the V L comprises LC-CDR1, 2, and 3 of the anti-ILT3 antibody disclosed herein or an embodiment wherein one or more of HC-CDR1, 2, and 3 has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof. In a further embodiment, the V H and / or V L variable region framework contains 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

[0289] In certain aspects, the present invention provides a nucleic acid molecule encoding V H wherein the VH comprises the HC CDR of the anti-ILT3 antibody disclosed herein or an embodiment wherein one or more of the three HC CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, and / or wherein V H and / or V L variable region framework contains 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof, and a nucleic acid molecule encoding V L wherein the V L comprises the LC CDR of the anti-ILT3 antibody disclosed herein or an embodiment wherein one or more of the three LC CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, and / or wherein V H and / or V L variable region framework contains 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

[0290] Mammalian cell lines that can be used as hosts for expressing antibodies or fragments are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include in particular Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (such as HepG2), A549 cells, 3T3 cells, human embryonic kidney 293 (HEK-293) cells and many other cell lines. Particularly preferred cell lines are selected by determining which cell lines have high expression levels. Other cell lines that can be used are insect cell lines such as Sf9 cells, amphibian cells, bacterial cells, plant cells, filamentous fungal cells (such as Trichoderma reesei) and yeast cells (such as Saccharomyces cerevisiae or Pichia pastoris). In certain aspects, the host cell can be a prokaryotic host cell such as Escherichia coli.

[0291] When a recombinant expression vector, which includes a nucleic acid molecule encoding a heavy chain or an antigen-binding portion or fragment thereof, the light chain and / or antigen-binding fragment is introduced into a host cell, the antibody is produced by culturing the host cell under conditions and for a time sufficient to allow expression of the antibody in the host cell, or more preferably, to secrete the antibody into the culture medium in which the host cell is growing. The antibody can be recovered from the culture medium and further purified or processed to produce the antibody of the invention.

[0292] In a specific aspect, the host cell is transfected with an expression vector comprising a nucleic acid molecule encoding an HC and an LC, wherein the HC comprises the HC-CDR3 of the anti-ILT3 antibody disclosed herein or an embodiment thereof in which the HC-CDR3 has one, two or three amino acid substitutions, additions, deletions or combinations thereof. In a further embodiment, the HC and / or LC variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof.

[0293] In a specific aspect, the host cell is transfected with an expression vector comprising a nucleic acid molecule encoding an HC and an LC, wherein the HC comprises the HC-CDR1, 2 and 3 of the anti-ILT3 antibody disclosed herein or an embodiment thereof in which one or more of HC-CDR1, 2 and 3 has one, two or three amino acid substitutions, additions, deletions or combinations thereof, and wherein the LC comprises the LC-CDR1, 2 and 3 of the anti-ILT3 antibody disclosed herein or an embodiment thereof in which one or more of HC-CDR1, 2 and 3 has one, two or three amino acid substitutions, additions, deletions or combinations thereof. In a further embodiment, the HC and / or LC variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof.

[0294] In a specific aspect, a host cell is transfected with a first expression vector and a second expression vector, the first expression vector comprising a nucleic acid molecule encoding an HC, the HC comprising an HC CDR of an anti-ILT3 antibody disclosed herein or an embodiment wherein one or more of the three HCCDRs thereof has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, and / or wherein the HC variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof, the second expression vector comprising a nucleic acid molecule encoding an LC, the LC comprising an LC CDR of an anti-ILT3 antibody disclosed herein or an embodiment wherein one or more of the three LC CDRs thereof has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, and / or wherein the LC variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

[0295] In a particular aspect, a host cell is transfected with an expression vector comprising a nucleic acid molecule encoding V H and V L wherein V H comprises at least an HC-CDR3 of an anti-ILT3 antibody disclosed herein or an embodiment thereof, wherein the HC-CDR3 has 1, 2, or 3 amino acid substitutions, additions, deletions, or combinations thereof. In a further embodiment, the V H and / or V L variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

[0296] In a specific aspect, a host cell is transfected with an expression vector comprising a nucleic acid molecule encoding V H and V L wherein V H comprises HC-CDR1, 2, and 3 of an anti-ILT3 antibody disclosed herein or an embodiment wherein one or more of HC-CDR1, 2, and 3 has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, and wherein V L comprises LC-CDR 1, 2, and 3 of an anti-ILT3 antibody disclosed herein or an embodiment wherein one or more of LC-CDR1, 2, and 3 has 1, 2, or 3 amino acid substitutions, additions, deletions, or combinations thereof. In a further embodiment, the V H and / or V L variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

[0297] In certain aspects, a host cell is transfected with a first expression vector and a second expression vector, the first expression vector comprising a nucleic acid molecule encoding V H , the V H comprising at least an HC CDR of an anti-ILT3 antibody disclosed herein or an embodiment wherein one or more of the three HC CDRs thereof has one, two or three amino acid substitutions, additions, deletions or combinations thereof, and / or wherein the V H variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof, the second expression vector comprising a nucleic acid molecule encoding V L , the V L comprising at least an LC CDR of an anti-ILT3 antibody disclosed herein or an embodiment wherein one or more of the three LC CDRs thereof has one, two or three amino acid substitutions, additions, deletions or combinations thereof, and / or wherein the V L variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof.

[0298] In certain embodiments, the HC and LC or V H and V L are expressed as a fusion protein, wherein the N-termini of the HC and LC are fused to a leader sequence to facilitate transport of the antibody through the secretory pathway. Examples of leader sequences that can be used include MSVPTQV L GLLLLWLTDARC (SEQ ID NO:12) or MEWSWVFLFFLSVTTGV H S (SEQ ID NO:11).

[0299] The invention further provides a plasmid or viral vector comprising a nucleic acid molecule encoding an anti-ILT3 antibody disclosed herein or an antigen-binding fragment thereof. The invention further provides a plasmid or viral vector comprising a nucleic acid molecule encoding an HC of an anti-ILT3 antibody disclosed herein or an antigen-binding fragment thereof or an embodiment wherein one or more of the three CDRs thereof has one, two or three amino acid substitutions, additions, deletions or combinations thereof, and / or wherein the HC variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof, and a nucleic acid molecule encoding an LC of an anti-ILT3 antibody disclosed herein or an antigen-binding fragment thereof or an embodiment wherein one or more of the three CDRs thereof has one, two or three amino acid substitutions, additions, deletions or combinations thereof, and / or wherein the LC variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitution positions, additions, deletions or combinations thereof.

[0300] The present invention further provides a plasmid or viral vector comprising a nucleic acid molecule encoding the HC of the anti-ILT3 antibody or antigen-binding fragment thereof disclosed herein, and a plasmid or viral vector comprising a nucleic acid molecule encoding the LC of the anti-ILT3 antibody or antigen-binding fragment thereof disclosed herein.

[0301] The present invention also provides a host cell comprising a plasmid or viral vector comprising a nucleic acid molecule encoding the HC of the anti-ILT3 antibody or antigen-binding fragment thereof disclosed herein or an embodiment of the anti-ILT3 antibody or antigen-binding fragment thereof disclosed herein, wherein one or more of the three CDRs have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, and / or wherein the HC variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof, and a plasmid or viral vector comprising a nucleic acid molecule encoding the LC of the anti-ILT3 antibody or antigen-binding fragment thereof disclosed herein or an embodiment of the anti-ILT3 antibody or antigen-binding fragment thereof disclosed herein, wherein one or more of the three CDRs have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, and / or wherein the LC variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof. In certain embodiments, the host cell is a CHO or HEK-293 host cell.

[0302] The present invention further provides a plasmid or viral vector comprising a nucleic acid molecule encoding the anti-ILT3 antibody or antigen-binding fragment thereof disclosed herein. The present invention further provides a plasmid or viral vector comprising a nucleic acid molecule encoding the V H or an embodiment of the anti-ILT3 antibody or antigen-binding fragment thereof, wherein one or more of the three CDRs have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, and / or wherein the V H framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof, and a nucleic acid molecule encoding the V L or an embodiment of the anti-ILT3 antibody or antigen-binding fragment thereof, wherein one or more of the three CDRs have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, and / or wherein the V L framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

[0303] The present invention further provides a plasmid or viral vector comprising a nucleic acid molecule encoding an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein or an antigen-binding fragment, and a plasmid or viral vector comprising a nucleic acid molecule encoding an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein or an antigen-binding fragment. H The present invention further provides a plasmid or viral vector comprising a nucleic acid molecule encoding an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein or an antigen-binding fragment, and a plasmid or viral vector comprising a nucleic acid molecule encoding an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein or an antigen-binding fragment. L The present invention further provides a plasmid or viral vector comprising a nucleic acid molecule encoding an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein or an antigen-binding fragment, and a plasmid or viral vector comprising a nucleic acid molecule encoding an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein or an antigen-binding fragment.

[0304] The present invention also provides a host cell comprising a plasmid or viral vector, which comprises a nucleic acid molecule encoding an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein or an embodiment of an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein, wherein one or more of the three CDRs have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, and / or wherein the variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof, and a plasmid or viral vector, which comprises a nucleic acid molecule encoding an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein or an embodiment of an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein, wherein one or more of the three CDRs have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, and / or wherein the variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof. In certain embodiments, the host cell is a CHO or HEK-293 host cell. H The present invention also provides a host cell comprising a plasmid or viral vector, which comprises a nucleic acid molecule encoding an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein or an embodiment of an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein, wherein one or more of the three CDRs have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, and / or wherein the variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof, and a plasmid or viral vector, which comprises a nucleic acid molecule encoding an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein or an embodiment of an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein, wherein one or more of the three CDRs have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, and / or wherein the variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof. In certain embodiments, the host cell is a CHO or HEK-293 host cell. H The present invention also provides a host cell comprising a plasmid or viral vector, which comprises a nucleic acid molecule encoding an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein or an embodiment of an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein, wherein one or more of the three CDRs have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, and / or wherein the variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof, and a plasmid or viral vector, which comprises a nucleic acid molecule encoding an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein or an embodiment of an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein, wherein one or more of the three CDRs have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, and / or wherein the variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof. In certain embodiments, the host cell is a CHO or HEK-293 host cell. L The present invention also provides a host cell comprising a plasmid or viral vector, which comprises a nucleic acid molecule encoding an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein or an embodiment of an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein, wherein one or more of the three CDRs have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, and / or wherein the variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof, and a plasmid or viral vector, which comprises a nucleic acid molecule encoding an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein or an embodiment of an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein, wherein one or more of the three CDRs have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, and / or wherein the variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof. In certain embodiments, the host cell is a CHO or HEK-293 host cell. L The present invention also provides a host cell comprising a plasmid or viral vector, which comprises a nucleic acid molecule encoding an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein or an embodiment of an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein, wherein one or more of the three CDRs have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, and / or wherein the variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof, and a plasmid or viral vector, which comprises a nucleic acid molecule encoding an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein or an embodiment of an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein, wherein one or more of the three CDRs have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, and / or wherein the variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof. In certain embodiments, the host cell is a CHO or HEK-293 host cell.

[0305] The anti-ILT3 antibody or its antigen-binding fragment can be recovered from the culture medium using standard protein purification methods. In addition, a variety of known techniques can be used to enhance the expression of the antibody (or other parts thereof) of the present invention from the production cell line. For example, the glutamine synthetase gene expression system (GS system) is a commonly used method to enhance expression under certain conditions.

[0306] Typically, a glycoprotein produced in a particular cell line or transgenic animal will have a glycosylation pattern that is characteristic of the glycoprotein produced in the cell line or transgenic animal (see, for example, Croset et al., J. Biotechnol. 161: 336-348 (2012)). Thus, the specific glycosylation pattern of the antibody will depend on the particular cell line or transgenic animal used to produce the antibody. However, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein constitute the present invention independent of the glycosylation pattern the antibody may have.

[0307] The following examples are intended to facilitate further understanding of the present invention.

[0308] General methods

[0309] Sambrook, Fritsch and Maniatis (1982 & 1989 2nd Edition, 2001 3rd Edition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA) describe standard methods in molecular biology. Standard methods also appear in Ausbel, et al. (2001) Current Protocols in Molecular Biology, Vols. 1 - 4, John Wiley and Sons, Inc. New York, NY, which describe cloning and DNA mutagenesis in bacterial cells (Volume 1), cloning in mammalian cells and yeast (Volume 2), glycoconjugates and protein expression (Volume 3) and bioinformatics (Volume 4).

[0310] Methods for protein purification are described, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization (Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and glycosylation of proteins are described (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, MO; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391). Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described (Coligan, et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques for characterizing ligand / receptor interactions are available (see, e.g., Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

[0311] Monoclonal antibodies, polyclonal antibodies, and humanized antibodies can be prepared (see, e.g., Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ. Press, New York, NY; Kontermann and Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 139-243; Carpenter, et al. (2000) J. Immunol. 165:6205; He, et al. (1998) J. Immunol. 160:1029; Tang et al. (1999) J. Biol. Chem. 274:27371-27378; Baca et al. (1997) J. Biol. Chem. 272:10678-10684; Chothia et al. (1989) Nature 342:877-883; Foote and Winter (1992) J. Mol. Biol. 224:487-499; U.S. Pat. No. 6,329,511).

[0312] Alternative humanization methods use human antibody libraries displayed on phage or in transgenic mice (Vaughan et al. (1996) Nature Biotechnol. 14:309-314; Barbas (1995) Nature Medicine 1:837-839; Mendez et al. (1997) Nature Genetics 15:146-156; Hoogenboom and Chames (2000) Immunol. Today 21:371-377; Barbas et al. (2001) Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Kay et al. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, San Diego, CA; de Bruin et al. (1999) Nature Biotechnol. 17:397-399).

[0313] Antibodies can be conjugated to, for example, small drug molecules, enzymes, liposomes, polyethylene glycol (PEG). Antibodies can be used for therapeutic, diagnostic, kit or other purposes and include, for example, antibodies conjugated to dyes, radioisotopes, enzymes or metals (such as colloidal gold) (see, e.g., Le Doussal et al. (1991) J. Immunol. 146:169-175; Gibellini et al. (1998) J. Immunol. 160:3891-3898; Hsing and Bishop (1999) J. Immunol. 162:2804-2811; Everts et al. (2002) J. Immunol. 168:883-889).

[0314] The method of flow cytometry can be used, which includes fluorescence-activated cell sorting (FACS) (see, e.g., Owens, et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2nd ed.; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ). Fluorescent reagents suitable for modifying nucleic acids, such as those used as diagnostic reagents for example, include nucleic acid primers and probes, polypeptides, and antibodies (Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, OR; Sigma-Aldrich (2003) Catalogue, St. Louis, MO).

[0315] Standard methods for the histology of the immune system are described (see, e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY; Hiatt, et al. (2000) Color Atlas of Histology, Lippincott, Williams, and Wilkins, Phila, PA; Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, NY).

[0316] Software packages and databases are provided for determining, for example, antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments (e.g., see GenBank, Vector Suite (Informax, Inc, Bethesda, MD); GCG Wisconsin Package (Accelrys, Inc., San Diego, CA); (TimeLogic Corp., Crystal Bay, Nevada); Menne, et al. (2000) Bioinformatics 16:741-742; Menne, et al. (2000) Bioinformatics Applications Note 16:741-742; Wren, et al. (2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne (1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res. 14:4683-4690).

[0317] Purity determination: Size-exclusion ultra performance liquid chromatography (SE-UPLC) or (SEC) was performed on an ACQUITY UPLC H-Class system. The column used was an ACQUITY UPLC Protein BEH SEC column (part number 186005225, 1.7 µm, 200 Å, 4.6 mm × 150 mm) from Waters (Milford, Massachusetts). The column temperature was 25 °C, and 10 µL of the sample at 1 mg / mm was injected using a system flow rate of 0.5 mL / min. The mobile phase used was 100 mM sodium phosphate, 200 mM sodium chloride, and 0.02% sodium azide, pH 7.0. Data were quantified at 214 and 280 nm and analyzed using Empower3 software. A BEH200 SEC Protein Standard Mix (part number 186006518) from Waters (Milford, Massachusetts) was used and injected at a dose of 10 µg to measure USP Resolution, theoretical plates, and tailing.

[0318] NANO-DSF (a method for determining protein stability by modified differential scanning fluorescence, using the intrinsic tryptophan or tyrosine fluorescence): The temperature midpoint Tm of the thermal unfolding curve and the midpoint Tagg of the thermal aggregation curve were determined by NANO-DSF using a Prometheus NT.48 differential scanning fluorometer (Nanotemper Technologies) controlled by PRTherm Control v2.0.4 software. The excitation power was 40%, and the temperature was increased from 20 °C to 95 °C at a rate of 1 °C / min. Tm and Tagg were automatically measured. Samples were prepared by diluting to 1 mg / mL in 20 mM sodium acetate buffer pH 5.5 and aspirated into Prometheus glass capillaries (PR-L002) by capillary action.

[0319] Capillary isoelectric focusing (cIEF): cIEF was performed on an iCE3 system obtained from Protein Simple (San Jose, CA) using iCE CFR software 4.1.1 for instrument control and data analysis. The cIEF cartridges used were Fc-coated (Protein Simple, 101701) and prepared according to the manufacturer's instructions. The preparation consisted of a 200 μL sample of 40 μg analyte and 1% v / v 3-10 Pharmalyte, 0.5% v / v 8-10.5 Pharmalyte, 0.5% v / v 5-8 Pharmalyte (GE Healthcare), 37.5% v / v 8.0 M urea (Sigma-Aldrich), 35% v / v 1% methylcellulose, and 1 μL each of 5.85 and 9.22 pI markers (Protein Simple). The sample was injected for 60 seconds. The isoelectric focusing parameters were 1500 V for 1 minute and 3000 V for 8 minutes. The pI was automatically measured using an internal pI marker as a two-point calibration standard. The calibrated data was further analyzed and quantified by converting it to Empower format using Empower3 and analyzed.

[0320] Example 1

[0321] Hybridoma clone 52B8 was identified by standard mouse and rat immunizations and hybridoma selections. Typically, in a standard four-week footpad immunization, Balb / C mice or rats were immunized with human ILT3-HIS recombinant protein to generate a hyperimmune response. The bulk lymphocytes from the draining lymph nodes were electrofused with the P3 myeloma fusion partner to generate immortalized hybridomas. The hybridoma supernatants were screened for human CHO-human ILT3 cells in a primary cell-based ELISA binding assay. The CHO parental, CHO-ILT3 SNP, CHO-rhesus ILT3, CHO-ILT5, CHO-ILT8, and CHO-ILT11 cells were secondarily screened in a cell-based ELISA format (see Example 2). Subcloning was performed by limiting dilution on ILT3-specific and rhesus-positive hybridoma cells. The subclones were amplified to produce purified proteins to enable additional tests for Biacore analysis and functional screening. Table 5 shows 10 hybridoma clones that produce antibodies that group together and have high affinity for human ILT3, as shown by CELISA and Biacore performed as disclosed in Examples 2 and 4, respectively.

[0322]

[0323] Table 6 shows the amino acid sequences of the heavy and light chain variable domains of the mAbs obtained from the above clones.

[0324]

[0325]

[0326] To ultimately guide the selection of lead antibodies, the antibodies were further analyzed and re-evaluated in a panel of biological, biophysical, and physicochemical assays. Finally, the antibodies were tested in an in vivo biological evidence tumor regression study using humanized mice challenged with human SKMEL5 melanoma.

[0327] Example 2

[0328] Selectivity of various anti-ILT3 antibodies

[0329] A cell-based ELISA (cELISA) was used to demonstrate the selectivity of various parental anti-ILT3 antibodies and the humanized anti-ILT3 monoclonal antibody 9B11 (disclosed in U.S. Patent No. 7,777,008, which has the amino acid sequences of SEQ ID NO:33 (light chain) and SEQ ID NO:34 (heavy chain)) shown in Table 5.

[0330] The binding of mouse anti-human ILT3 antibody to human ILT3 and its cross-reactivity with macaque ILT3, human ILT5, human ILT7, human ILT8, and human ILT11-expressing CHO-K1 cells were tested using a cell-based ELISA format. CHO-K1 cells were seeded in 96-well tissue culture plates in 50 μL of DMEM / F12, 10% BCS, and gentamicin (CHO-K1 medium). Cells were plated at 2×10 4 cells / well two days before the assay or 4×10 4 cells / well one day before the assay. The medium was removed from the wells before adding the test samples. The purified antibodies were serially diluted in CHO-K1 medium and then added to the CHO-K1 plates. The samples were incubated at room temperature for 30 - 60 minutes and the plates were washed 3 times with PBS / 0.5% Tween-20 using the cell washing program on a Biotek EL405xSelect CW plate washer. Binding was detected using a secondary antibody, HRP-conjugated goat anti-mouse IgG (Southern Biotech cat#1031-05) added at a 1:2000 dilution in CHO-K1 medium and incubated at room temperature for 30 - 60 minutes. The assay plates were washed as above and developed with TMB and terminated with TMB stop solution (KPL catalog number 50-85-06). Absorbance was measured at 450 nm - 620 nm. Mouse IgG1 (MIgG1) was used as a control.

[0331] The results are shown inFigure 1A , 1B , 1C, 1D, and 1E. These figures show that representative antibodies from clones p40B5, p49C6, and p52B8 are specific for ILT3 and do not cross-react with or bind to ILT5, ILT7, ILT8, and ILT11. Antibodies from clones p49C6 and p52B8, as well as antibodies from other clones, are able to bind rhesus ILT3. Clone p52B8 was selected for in vivo characterization based on (1) its high affinity for human ILT3, (2) lack of binding to other ILT family members, and (3) cross-reactivity with rhesus ILT3.

[0332] Example 3

[0333] The parental mouse 52B8 heavy chain (V H ) and light chain (V L ) variable domain sequences were compared to human germline sequences. Human framework sequences that were closely homologous to the framework of the mouse antibody were selected.

[0334] The mouse V H domain of the mouse anti-human ILT3 mAb clone 52B8 scored highly against human heavy chain germline 3-07 in subgroup III and JH4 of the J region. Based on structural considerations, two framework substitutions (R87K and A97G) were introduced to maintain equivalent binding to the parental antibody. The mouse V L domain of this antibody clone scored highly against human light chain germline 1-O2 in kappa subgroup I. The mouse 52B8 CDRs were engineered onto the variable light chain sequences of 1-O2 and JK2 in the J region. Based on structural considerations, three framework substitutions (M4L, S64A, and G72R) were introduced.

[0335] To generate humanized variants, the humanized V H sequences were cloned into a vector encoding the human IgG4 S228P heavy chain constant domain, and the humanized V L domain was cloned into a vector encoding the kappa light chain constant domain. A total of two humanized V H s (V H 1 and V H 2) and eight humanized V L s were designed. Computational sequence and structural analysis of mouse 52B8 revealed six potential "hot spots" on the molecule: two potential oxidation sites in V H -CDR2 (M64) and V H -CDR3 (W101), one potential isomerization site in V H -CDR2 (D62), and V L- A potential deamidation site in CDR1 (N34), V L - CDR1 (D30) and V L - Two potential isomerization sites in CDR2 (D59). M64 was modified to V64 or L64, which maintained good physicochemical properties and binding / function.

[0336] Figure 2A Tables are provided that show data characteristics regarding the binding affinity, isoelectric point, monomeric species purity, and thermal stability measurements of the designed humanized variants. Biacore was used to measure the binding affinity, cIEF was used to measure the pI, purity was determined by SE - UPLC, and Tm and Tgg were determined by Nano - DSF. Figure 2B Shows the relationship between SEC purity and melting temperature of various humanized light - chain variants. The data were plotted as values obtained from each of eight humanized light - chain variants, indicating that V L 5 has the highest purity and thermal stability both. Based on Figure 2A and Figure 2B data, V L 5 was selected for the light chain.

[0337] A preliminary study was conducted on the humanized V H 1 M64V / V L 5 produced in transient CHO cells. Forced deamidation conditions of 50 °C incubation and high - pH stress were performed simultaneously on unformulated humanized 52B8 V H 1 M64V / V L 5, showing deamidation of LCN34 in V L - CDR1 (4.0% and 7.2% respectively), and oxidation of W101 in HC - CDR3 at 1× light stress exposure of 15.4%. Substitution of N34 with Q34 was evaluated for maintaining binding affinity to human and cynomolgus ILT3 using Biacore SPR analysis and functional activity using DC TNFα production assay; however, substitution of the W101 residue resulted in a significant loss of binding as determined by Biacore SPR assay.

[0338] In summary, humanized 52B8 is an anti - ILT3 mAb (52B8 V H 1 M64V / V L 5 N34QIgG4 S228P / κ), containing one framework substitution (M4L) in V L and one framework substitution (A97G) in V H .

[0339] Example 4

[0340] The binding kinetics and affinity of anti-human ILT3 clones to human or cynomolgus ILT3-His-tagged recombinant proteins were measured by surface plasmon resonance using a Biacore T200 system (GE Healthcare, Piscataway, NJ). HBS-EP+ buffer (BR-1006-69) was used as the running buffer. Anti-human Fc antibody (Human Fc Capture Kit, BR100839, GE Healthcare) was immobilized on all four flow cells of a Series S CM5 sensor chip (BR100530 or 29149603, GE Healthcare) by amine coupling chemistry according to the manufacturer's instructions. Flow cell 1 was used as a reference for background subtraction and not for capture. The anti-human ILT3 antibodies listed above (diluted to 1 μg / mL in HBS-EP+ buffer) were injected for 10 seconds at 10 μL / mL onto the anti-human Fc capture surface in flow cells 2, 3, and 4, which resulted in antibody capture levels in the range of 60 - 70 RU. A two-fold dilution series of human or cynomolgus ILT3-His protein ranging from 20 nM to 0.31 nM and two zeros (HBS-EP+) were injected for 180 seconds at 50 μL / mL onto the reference and capture antibody surfaces for binding, followed by 600 seconds of dissociation. After each injection cycle, all four flow cells were regenerated with a 30-second second injection of 3M MgCl 2 solution. In the Biacore T200 evaluation software (version 2.0), the sensorgram with reference subtracted was fitted to a 1:1 Langmuir binding model to determine the association (ka) and dissociation (kd) rate constants and the equilibrium dissociation constant KD (= kd / ka).

[0341] Table 7 summarizes the binding kinetics and affinity of the anti-human ILT3 antibody to recombinant human or cynomolgus ILT3.

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349] Example 5

[0350] Chimeric anti-ILT3 52B8 murine V H / human IgG4 (S228P): murine V L / human κ (“c58B8”; mAb73) Epitope mapping by hydrogen-deuterium exchange (HDX) mass spectrometry

[0351] The contact regions of the antibody with the extracellular domain of human ILT3 were determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis. HDX-MS measures the incorporation of deuterium into the amide backbone of the protein, and the change in this incorporation is affected by the solvent exposure of the hydrogen. The deuterium exchange levels in samples of the individual antigen and the bound antibody were compared to determine the regions on the extracellular domain of ILT3 that could be in contact with the antibody. The extracellular domain of human ILT3 with a C-terminal His tag (human ILT3-His) has the amino acid sequence shown in SEQ ID NO:1.

[0352] The His-tagged extracellular domain of human ILT3-His was pre-incubated with the antibody c58B8 (mAb 73), which is chimeric anti-ILT3 52B8 murine V H M64V / human IgG4 (S228P): murine V L / human κ, which comprises an HC having the amino acid sequence of SEQ ID NO:113 and an LC having the amino acid sequence shown in SEQ ID NO:116, and then incubated in deuterium buffer. The human ILT3-His and the antibody were buffer-exchanged to PBS pH7.4 using a 3k MWCO centrifugal column. Human ILT3-His (80 pmol / μL) was mixed with an equal volume of the antibody (40 pmol / μL), or as an unbound control, PBS pH7.4. Before starting the labeling experiment, the samples of the bound antibody and the unbound control were incubated at room temperature for 1 hour.

[0353] To deuterate the sample, 2 μL of the sample was mixed with 25 μL of PBS in deuterium oxide at pH 7.6. The labeling time points were 30, 300, 3000, 6000 or 12000 seconds. After the set time, 25 μL of the labeling mixture was added to 30 μL of cold quenching buffer (8 M urea, 150 mM TCEP). The quenched sample was incubated at 1.5 °C for 2 minutes. 53 μL was then injected into the column cooling chamber, where the sample was passed through a pepsin / protease XIII column and the resulting peptides were loaded onto a capture column. Three minutes later, the analytical gradient and the mass spectrometer were started. A fully deuterated sample was generated by incubating 2 μL of human ILT3-His with 108 μL of deuterated denaturing buffer (4 M urea, 150 mM TCEP in 99.5% deuterium oxide). The sample was incubated overnight at 37 °C. Then 55 μL was directly injected into the column chamber and data were acquired.

[0354] LC-MS / MS data of the unlabeled sample were acquired and searched before deuteration to verify successful digestion of the protein and generate a peptide list. The database was searched using the Proteome Discoverer 1.4 and SEQUEST HT search algorithms (ThermoFisher Scientific). The protein database used was the human ILT3-His sequence linked to the Saccharomyces cerevisiae database.

[0355] After labeling, 55 μL aliquots of the sample were applied to a NovaBioAssays pepsin / protease XIII column, followed by chromatography on a Waters CSH C18 guard column and a Waters CSH C18 1x50 mm analytical column in a loading buffer containing 2% acetonitrile, 0.1% TFA. Deuterium incorporation into the extracellular domain of human ILT3-His was measured by mass spectrometry. Quenching: 8 M urea, 150 mM TCEP; Labeling buffer: PBS, pH 7.6; Blank buffer: PBS, pH 7.4. The mass spectrometer was a ThermoScientific Orbitrap-Elite. To measure the deuterated sample, the mass spectrometer was set to acquire one full scan MS data in the orbitrap at a resolution of 120,000, a target ion number of 1E6 and a maximum ion injection time of 500 milliseconds. To obtain MS / MS data for peptide identification, the mass spectrometer was set to acquire one full scan spectrum at a resolution of 120,000 and then ten data-dependent MS / MS spectra in the ion trap.

[0356] The liquid chromatography system used was a Waters nano ACQUITY for analytical column gradients and a Waters 515 isocratic pump for sample digestion and loading. For sample digestion and loading, the buffer used was 2% acetonitrile and 0.1% trifluoroacetic acid, with a flow rate of 100 μL / min. For the analytical gradient, the buffers were buffer A) 0.1% formic acid in water, and buffer B) 0.1% formic acid in acetonitrile. The gradient was from 2% B to 36% B in 10 minutes at 40 μL / min, then washed with 80% B for 1.5 minutes, and then re-equilibrated at 2% B for 3 minutes. The column was then washed by cycling the gradient three times between 2% and 80% B, 1 minute per step, and then finally equilibrated at 2% B for 5 minutes. The capture column was a Waters Vanguard C18 BEH 1.7 μm guard column, and the analytical column was a Waters C18 BEH300, 1.7 μm 1×50 mm column.

[0357] Deuterium-labeled sample processing was done via a Leaptec H / D-X PAL system. The labeled sample tray temperature was set to 25 °C, the quench tray temperature was set to 1.5 °C, and the trap and analytical column chamber were set to 1.5 °C. The immobilized pepsin column (pepsin / protease XIII column NBA2014002, 2.1x30 mm, NovaBioAssay) was kept outside the column chamber at room temperature.

[0358] The deuterium-labeled differential thermogram of the antibody-bound human ILT3-His amino acid residues is shown in Figure 3A ... HDX mass spectrometry indicates that the antibodies disclosed herein and other antibody families that cross-compete with the antibody bind to an epitope that comprises or consists of at least one amino acid from one or more of amino acid residues 18 - 23 (ISWGNS; SEQ ID NO:3), 64 - 69 (IPSMTE; SEQ ID NO:4), 96 - 101 (MTGAYS; SEQ ID NO:5), 124 - 131 (QSRSPMDT; SEQ ID NO:6), 152 - 159 (AQQHQAEF; SEQ ID NO:7), and 184 - 187 (LLSH; SEQ ID NO:8) of ILT3. Figure 3B The first and second views of a three-dimensional surface structure model of the extracellular domain of human ILT3 are shown, showing the protected amino acid residues. These protected amino acid residues comprise a split or discontinuous epitope spanning the boundary between the D1 and D2 domains of the extracellular domain. Figure 3CIt is a strip chart showing the positions of epitopes on the extracellular domain of human ILT3. The black residues are protected from antibody labeling. The white residues show no change in terms of labeling, and the dark gray residues have no data obtained from them. The deuterium labeling difference per residue on average was mapped onto the crystal structure of ILT3 (Cheng et al., “Crystal structure of leukocyte Ig-like receptor LILRB4 (ILT3 / LIR-5 / CD85k): a myeloid inhibitory receptor involved in immune tolerance.” J Biol Chem 286:18013 - 25 (2011)).

[0359] Similar HDX mapping experiments were performed using antibodies ZM4.1, DX439, DX446, and 9B11. Antibody ZM4.1 is commercially available from ThermoFisher Scientific, Carlsbad, CA or BioLegend, San Diego, CA. Antibodies DX439 and DX446 have been disclosed in WO2018089300, and antibody 9B11 has been disclosed in US Patent 7,777,008. Among these antibodies, only antibody ZM4.1 was observed to bind to an epitope that partially overlaps with the epitope bound by the antibodies of the present invention. However, the cross - blocking study showed that antibody ZM4.1 did not cross - block the binding of the antibodies of the present invention. Figure 3D , 3E, 3F, and 3G show the heat maps of the binding of antibodies ZM4.1, DX439, DX446, and 9B11 to human ILT3.

[0360] Example 6

[0361] Chimeric anti - ILT3 52B8 mouse V H / human IGG4(S228P): mouse V L / human κ (“c58B8”; mAb 73) pharmacokinetics in NSG mice

[0362] The pharmacokinetics of chimeric anti - ILT3 52B8 mouse V H / human IgG4(S228P): mouse V L / human κ (c85B8; mAb 73) were evaluated in Panc08.13 human - NSG mouse model and SK - MEL - 5 human CD34+ - NSG mouse model.

[0363] SK-MEL-5 is a human melanoma-derived cell line that can grow as subcutaneous tumors. Panc08.13 is a human pancreatic cancer-derived tumor cell line. The Panc 08.13 human-NSG model has shown sensitivity to pembrolizumab and ipilimumab treatment. Compared to the Panc 08.13 model, the SK-MEL-5 model has a robust and diverse myeloid infiltration in the tumor. Both models show increased ILT3 expression on human CD14+ myeloid cells in the tumor and spleen.

[0364] An ECL-based target capture immunoassay was used to quantify antibodies in humanized mouse plasma. The assay was established using biotinylated recombinant ILT3 as the capture reagent and a sulfoTAG-labeled mouse anti-huIgG (Fc specific) from Southern Biotech (cat#9190-01) as the detection reagent. Calibrators and QCs were prepared in pure C57BL / 6 plasma and diluted 100-fold when tested in the plate. The assay has been qualified, with the LLOQ of the assay determined to be 40 ng / mL and the MRD being 100.

[0365] In the Panc08.13 hu-NSG mouse model, the antibody was administered IP at 20 mg / kg once a week for the first three doses and two weeks after the third dose for the fourth dose, with or without pembrolizumab (5 mg / kg). Blood samples were collected before the third dose (Ctrough) and 24 hours after the third dose (Cmax). Terminal blood samples were also collected on days 5 and 6 after the fourth dose. In the SK-MEL-5 huCD34+-NSG mouse model, the antibody was administered IP at 2 and 20 mg / kg weekly. Blood samples were collected before the third dose (Ctrough) and 24 h after the third dose (Cmax). Terminal blood samples were also collected on days 3 and 7 after the third dose. Free (unbound) antibody concentrations were determined by an antigen capture assay.

[0366] Pharmacokinetic parameters were generated from historical IgG4 antibody data (intravenous bolus administration of 1, 3, 10, 30 mg / kg of humanized IgG4 antibody in C57BL / 6J mice), and the Phoenix NLME.PK curves for the antibodies at the studied doses were simulated based on the generated pharmacokinetic parameters.

[0367] PK analysis of the historical IgG4 antibody data showed a linear relationship between AUC and the studied doses (see Figure 4)。Under the assumptions of linear PK of c52B8 across different test doses, no PK differences between different mouse strains, rapid absorption after IP administration of the antibody, and 100% bioavailability, the PK curves of the studied doses of c52B8 were simulated based on historical IgG4 antibody data. The results showed that in both the Panc08.13 human-NSG model and the SK-MEL-5 huCD34+-NSG model, the simulated curves at 20 mg / kg followed the observed c52B8 concentrations.

[0368] Example 7

[0369] The anti-ILT3 monoclonal antibody activates dendritic cells and reduces the inhibitory capacity of myeloid-derived suppressor cells (MDSCs)

[0370] Freeze, thaw, and isolate human PBMCs from fresh leukocytes and purify CD14+ monocytes by negative selection. The purified cells were cultured with GM-CSF (1000 U / mL) and IL4 (1000 U / mL) for 5 days. Then, these immature DCs were further cultured for 42 hours in the presence of IL-10 (50 ng / mL) and LPS (1 μg / mL) with or without anti-ILT3 antibody. TNFα was measured in the culture supernatant.

[0371] Titration experiments showed that c52B8, when added during the polarization step, caused a dose-dependent increase in TNFα secretion in the medium, while control IgG4 did not (the control was a variant of a commercially available antibody against RSV, trade name Synagis) ( Figure 5A )。The antibody concentration (EC50) required to produce a half-maximal increase in TNFα levels was approximately 1.9 ng / mL. For the V H and V L of p58B8, there was no difference for chimeric variants fused to the Fc of a human IgG1 framework (mAb 78) or a human IgG1 framework with an N297A mutation (mAb 76). These data indicate that in this assay, Fc receptor binding does not play any role in functional activity. Independence from Fc receptor binding controls the activation mechanism in this assay by ruling out the possibility that DCs become activated by recognizing other DCs in the culture decorated with the antibody, which would be an ILT3-independent mechanism.

[0372] Figure 5B and 5C Shown in two donors for c52B8 (mAb 73) and humanized anti-ILT3 mAb 52B8 V H 1M64 V / V LThere was no significant difference in functional activity between 5 N34Q IgG4 S228P / κ (mAb 46). As shown, addition of antibody c52B8 during DC polarization, but not during T cell priming, enabled DCs to better activate T cells to proliferate, similar to DCs intolerant to IL10. When antibody c52B8 was added during T cell priming, but not during DC polarization, T cells were better able to respond to subsequent restimulation. After humanization, variants that retained binding comparable to the chimeric were tested in the same assay and found to be active, with no meaningful differences in potency between them. These data suggest that the data generated with c52B8 are representative of what would be obtained if humanized mAb 46 were used.

[0373] Example 8

[0374] Anti-ILT3 antibodies reduce the inhibitory capacity of myeloid-derived suppressor cells (MDSCs)

[0375] Without ascribing to any particular theory or hypothesis, we postulate that the response of productive T cells to tumors can in some cases be limited by the presence of immature and inhibitory myeloid cells. These cells express ILT3, and we postulate that ILT3 functions in an inhibitory manner to maintain an immature state characterized by low HLA-DR expression, IL-10 production, and efficient inhibition of T cell activation and proliferation. A model based on in vitro co-culture of human PBMCs with SKMEL5 tumor cells was established, and then MDSCs were purified and tested for their ability to inhibit the proliferation of autologous CD8+ T cells, enabling exploration of this aspect of ILT3 biology. This example shows that c52B8 and humanized 52B8 (mAb 46) can impair the acquisition (or maintenance) of the T cell inhibitory phenotype.

[0376] To generate MDSCs, healthy human PBMCs were cultured with SKMEL5 cells and 20 ng / mL of GM-CSF for 7 days. CD33+ cells were collected by positive antibody-based magnetic bead selection and then co-cultured with purified autologous CD8+ T cells at the indicated ratios in the presence of polyclonal stimulants for 3 days. In both the co-culture and T cell inhibition steps, the cultures contained c52B8 (mAb 73), humanized 52B8 (mAb 46), or an isotype control antibody (1 μg / mL). T cell inhibition assays were performed at a T cell to MDSC ratio of 4:1, and the amount of interferon γ (INFγ) produced was measured.

[0377] Figure 6A and Figure 6BThe activities of humanized 52B8 and c52B8 in an MDSC model at a certain ratio of T cells to MDSCs are exemplified, where the effects of these antibodies are most obvious, indicating that these antibodies reduce the inhibitory ability of MDSCs in a comparable manner. These data further suggest that the data generated with c52B8 are representative of the data that would be found using humanized mAb 46.

[0378] Example 9

[0379] The anti-ILT3 antibody cC52B8 inhibits SK-MEL-5 tumor growth in SK-MEL-5 hu-NSG mice bearing subcutaneous SK-MEL-5 tumors

[0380] Systemic administration of c52B8 once a week to mice bearing established subcutaneous tumors provided inhibition of tumor growth ( Figure 7 ). Animals were randomly assigned to treatment groups on day 21 after implantation based on tumor volume and were dosed subcutaneously with 20 mg / kg c52B8 (mAb 73) or isotype control once a week starting on day 21. The data shown in the left panel are mean and std. error (nine per group). The right panel shows the individual animal tumor growth curves. Body weight loss in both the control group and the 52B8 group decreased to a similar extent. This study represents three independent studies.

[0381] In three independent studies, the degree of inhibition of tumor growth was consistent and similar and very similar to the effect of anti-ILT4. None of the other mechanisms tested to date (e.g., anti-PD-1, anti-ILT4, anti-CD27, anti-GITR) provided regression, leading us to speculate that tumor stasis may represent the basis of this model. This is significantly different from the murine syngeneic models commonly used for preclinical efficacy assays.

[0382] Example 10

[0383] Immune activation in SK-MEL-5 hu-NSG after c52B8 treatment

[0384] To understand the immune mechanisms mediating tumor efficacy, tumor-infiltrating immune cells were analyzed and measured sHLA-G levels in the blood were measured. Mice were treated with c52B8 (2 and 20 mg / kg i.p. QW). Based on C detected in mini-PK and simulations of historical studies max and C troughAntibody doses were selected horizontally. Blood samples were collected for PK, sHLA-G, and cytokine analysis. TIL analysis was performed using CyTOF to simultaneously detect 36 markers. Terminal tumor samples were fixed and used for human CD3+ T cell IHC analysis. A 30% tumor growth inhibition was observed in mice treated with 20 mPK 52B8. However, due to the large variability associated with the humanized tumor model, no statistically significant difference was detected. The modest tumor efficacy of 52B8 was associated with a modest decrease in tumor CD4+CD127-CD25+ T suppressor cells (21% compared to 14%) and blood sHLA-G levels, as well as an increase in the activation of T cells in the tumor (CD69 intensity, 14 compared to 23). As shown in Figure 8, no cytokine changes were detected for c52B8 treatment.

[0385] Example 11

[0386] Effect of anti-ILT3 antibody c52B8 in combination with pembrolizumab in the Panc08.13 hu-NSG model: Tumor efficacy and immune activation

[0387] Anti-ILT3 antibody c52B8 was evaluated in the Panc08.13 hu-NSG model. 52B8 used as a single agent showed little effect on tumor growth inhibition. When 52B8 was used in combination with pembrolizumab, one of five groups of humanized mice (five different human donors) had 50% tumor growth inhibition (TGI), and this TGI was associated with increased T cell activation and IFNγ production and decreased blood sHLA-G levels, as Figure 9A , Figure 9B , Figure 9C and Figure 9D shown.

[0388] Example 12

[0389] Effect of the combination of anti-ILT3 antibody 52B8 and pembrolizumab in the MDSC / T cell inhibition assay

[0390] In the MDSC / T cell inhibition assay, the humanized anti-ILT3 antibody 52B8 (mAb46) with and without pembrolizumab achieved increased T cell activity. When mAb 46 was used in combination with pembrolizumab, the effect was additive.

[0391] To generate MDSCs, healthy human PBMCs from a specific donor were cultured with SKMEL5 cells and 20 ng / mL of GM-CSF for seven days. The cultures were treated with 52B8 (1 μg / mL) or isotype control antibody (1 μg / mL). CD33+ cells were isolated using anti-CD33 magnetic microbeads and an LS column (Miltenyi Biotec, Germany) and then co-cultured with purified autologous CD8+ T cells at the specified ratios in the presence of a polyclonal stimulator for 3 days. Autologous CD8+ T cells were isolated from healthy human PBMCs using negative antibody-based magnetic bead selection (Stem Cell Technologies, Canada) and then co-cultured with CD33+ myeloid cells at a ratio of 8:1 (T cells:MDSCs) in 96-well plates for 2 days. In both the co-culture and T cell inhibition steps, the cultures contained humanized 52B8 (mAb 46) or isotype control antibody (IgG4) (1 μg / mL) alone or in combination with pembrolizumab (2 μg / mL). The total antibody concentration in each treatment was adjusted to 3 μg / mL with isotype control antibody. T cell proliferation was induced by polyclonal stimulatory anti-CD3 / CD28 beads and IL2. IFNγ levels were determined in the culture supernatants using MSD ELISA (Mesoscale Discovery, MD). T cell inhibition assays were performed with T cell to MDSC ratios of 4:1 or 8:1, and the amount of interferon γ (INFγ) produced was measured. The results are shown in Figures 10 - 14 in.

[0392] Figure 10 showed that in the MDSC / T cell inhibition assay, using MDSCs obtained from PBMCs from two different human donors (D00100385 and D001003507 respectively), the humanized anti-ILT3 antibody 52B8 (mAb 46) reduced the inhibitory capacity of MDSCs to a comparable extent to that of the chimeric anti-ILT3 antibody c52B8 (mAb 73; lot 26AVY).

[0393] Figures 11 - 14 showed that in the MDSC / T cell inhibition assay, (a) using MDSCs obtained from PBMCs from human donor D001003835, at T cell to MDSC ratios of 4:1 or 8:1 ( Figure 11 ); (b) using MDSCs obtained from PBMCs from human donor D001003180, at MDSC to T cell ratios of 4:1 or 8:1 ( Figure 12 ); (c) using MDSCs obtained from PBMCs from human donor D001003507, at T cell to MDSC ratios of 4:1 or 8:1 ( Figure 13);and using MDSC obtained from PBMC from human donors, at a T cell to MDSC ratio of 8:1( Figure 14 ), the humanized anti-ILT3 antibody 52B8 (mAb 46) in combination with pembrolizumab reduced MDSC inhibition of T cell activation at higher levels compared to each alone. The results are summarized in Tables 8 and 9. As Figures 10 - 13 shown in and Tables 8 and 9, combining the anti-ILT3 antibody 52B8 with pembrolizumab produced an additive effect that increased the activation of T cells relative to what was achievable with pembrolizumab or 52B8 alone. As shown, the increase in IFNγ for the combination ranged from 41% to 74% relative to other treatments. These results indicate that the combination of pembrolizumab and 52B8 does not result in an excessive or uncontrolled increase in T cell activation.

[0394]

[0395]

[0396]

[0397] Example 13

[0398] Effect of the combination of anti-ILT3 antibody 52B8 and pembrolizumab in the mixed lymphocyte reaction of polarized IL-10 DCs and allogeneic CD8+ T cells

[0399] In this example, the mixed lymphocyte reaction of IL-10 polarized human monocyte-derived dendritic cells and allogeneic CD8+ T cells was cultured for 4 days, and then interferon γ (IFNγ) in the culture supernatant was measured as a readout of T cell activation. In this experiment, the activities of pembrolizumab, 52B8, or a combination of both were compared to an isotype control antibody (IgG4 in both cases) in 9 allogeneic donor pairs.

[0400] Monocyte-derived dendritic cells (DC)-IL10 DCs from three CD14+ monocyte donors were differentiated for seven days (granulocyte-macrophage colony-stimulating factor (GMCSF) and IL4 for five days, then IL10 for two days), with and without IgG4 (lot 92ASJ), with and without 1 μg / mL of 52B8 (Lot 41BAB) to generate DC129, DC226, and DC196. CD8+ cells were isolated from three donors and a mixed leukocyte reaction (MLR) was set up in 96-well format at a DC:T cell ratio of 1:5 (30k DC vs 150k CD8+ T cells) from these three donors, with and without IgG4 (lot 92ASJ); cells were treated with and without 2 μg / mL of pembrolizumab (lot 42ASN). IgG4 or 52B8 was also added back to the MLR at 1 μg / mL. Ending with nine MLR pairs of IL10 DC:CD8+ T cells:

[0401] DC129 vs T30, T3788, and T3259

[0402] DC226 vs T30, T3788, and T3259

[0403] DC196 vs T30, T3788, and T3259

[0404] IFNγ supernatants were collected on day 4 and quantified using Meso Scale Discovery (MSD). Additional supernatant fractions were collected on day 5, cells were collected and stained for PD1 and PDL1 expression. Dendritic cells were stained on day 7 of differentiation (just before MLR setup). The MLR assay was performed with T cell staining of CD8+ T cells on day 5.

[0405] Figure 15 Results for all donor pairs were combined into one graph (each marker is a donor pair). As shown, combining 52B8 with pembrolizumab achieved reversal of T cell tolerance, resulting in a statistically significant increase in CD8+ T cell activation.

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

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[0421]

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[0428]

[0429] Although the present invention has been described with reference to the embodiments shown herein, it should be understood that the present invention is not limited thereto. Those having ordinary skill in the art and having access to the teachings herein will recognize other modifications and embodiments within its scope. Therefore, the present invention is limited only by the appended claims herein.

Claims

1. Use of an antibody or antigen-binding fragment that binds to human immunoglobulin-like transcript 3 (ILT3) in the preparation of a medicament for treating cancer, wherein the antibody or antigen-binding fragment comprises: Heavy chain (HC), wherein the heavy chain variable domain (V H ) comprises a heavy chain complementarity determining region (HC-CDR) 3 having an amino acid sequence selected from SEQ ID NO: 22, 49, 57, 65, 73, 81, 89, 97, and 105, or having an amino acid sequence that differs from an amino acid sequence selected from SEQ ID NO: 22, 49, 57, 65, 73, 81, 89, 97, and 105 by 3, 2, or 1 amino acid.

2. The use according to claim 1, wherein the antibody or antigen-binding fragment binds to an epitope on human ILT3, and the epitope comprises at least one amino acid from one or more of the amino acid sequences set forth in SEQ ID NO: 3, 4, 5, 6, 7, and 8.

3. Use of an antibody or antigen-binding fragment that binds to human immunoglobulin-like transcript 3 (ILT3) in the preparation of a medicament for treating cancer, wherein the antibody or antigen-binding fragment comprises: (a) heavy chain (HC) having a variable domain (V H ), which comprises a variable domain complementarity determining region (HC-CDR) 1 having the amino acid sequence shown in SEQ ID NO: 17, 47, 55, 63, 71, 79, 87, 95 or 103; an HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 18, 48, 56, 64, 72, 80, 88, 96 or 104; an HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 23, 49, 57, 65, 73, 81, 89, 97 or 105; and variants thereof in which one or more of the HC-CDRs have one, two or three amino acid substitutions, additions, deletions or combinations thereof; and (b) Light chain (LC), which has a variable domain (V L ), which comprises a variable domain complementarity determining region (LC-CDR) 1 having the amino acid sequence shown in SEQ ID NO: 27, 50, 58, 66, 74, 82, 90, 98 or 106; an LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 43, 51, 59, 67, 75, 83, 91, 99 or 107; an LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 44, 60, 68, 76, 84, 92, 100 or 108; and variants thereof in which one or more of the LC-CDRs have one, two or three amino acid substitutions, additions, deletions or combinations thereof.

4. The use according to claim 3, wherein (a) the HC-CDR1 has the amino acid sequence set forth in SEQ ID NO: 17; the HC-CDR2 has the amino acid sequence set forth in SEQ ID NO: 19, 20, or 21; and the HC-CDR3 has the amino acid sequence set forth in SEQ ID NO: 23; and (b) the LC-CDR1 has the amino acid sequence set forth in SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, 41, or 42; the LC-CDR2 has the amino acid sequence set forth in SEQ ID NO: 43; and the LC-CDR3 has the amino acid sequence set forth in SEQ ID NO:

44.

5. The use according to claim 4, wherein (a) the HC-CDR1 has the amino acid sequence set forth in SEQ ID NO: 17; the HC-CDR2 has the amino acid sequence set forth in SEQ ID NO: 20; and the HC-CDR3 has the amino acid sequence set forth in SEQ ID NO: 23; and (b) the LC-CDR1 has the amino acid sequence set forth in SEQ ID NO: 41; the LC-CDR2 has the amino acid sequence set forth in SEQ ID NO: 43; and the LC-CDR3 has the amino acid sequence set forth in SEQ ID NO:

44.

6. Use according to claim 3, 4 or 5, wherein said V H comprises a framework selected from human V H 1, V H 2, V H 3, V H 4, V H 5, and V H 6, and variants thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof; and, said V L comprises a framework selected from human V κ 1, V κ 2, V κ 3, V κ 4, V κ 5, V κ 6, V λ 1, V λ 2, V λ 3, V λ 4, V λ 5, V λ 6, V λ 7, V λ 8, V λ 9, and V λ 10, and variants thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof.

7. The use according to claim 3, 4, 5, or 6, wherein the antibody comprises an HC having a human IgG1, IgG2, IgG3, or IgG4 HC constant region or a variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native IgG1, IgG2, IgG3, or IgG4 isotype constant domain.

8. The use according to claim 6 or 7, wherein the antibody comprises an LC having a human κ or λ LC constant domain or a variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native human κ or λ light chain constant domain.

9. The use according to claim 5, wherein the antibody comprises: (i) V H , which has a framework selected from human V H 1, V H 2, V H 3, V H 4, V H 5 and V H 6 and a human IgG1 or IgG4 HC constant domain or a variant thereof that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native IgG1 or IgG4 isotype HC constant domain; and, (ii) V L which has a framework selected from human V κ 1, V κ 2, V κ 3, V κ 4, V κ 5, V κ 6, V λ 1, V λ 2, V λ 3, V λ 4, V λ 5, V λ 6, V λ 7, V λ 8, V λ 9 and V λ 10 and a human κ or λ LC constant domain or a variant thereof that contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof compared to the amino acid sequence of the native human κ or λ LC constant domain.

10. The use according to claim 6, wherein the antibody or antigen-binding fragment comprises V having the amino acid sequences set forth in SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 45 and SEQ ID NO: 46; SEQ ID NO: 53 and SEQ ID NO: 54; SEQ ID NO: 61 and SEQ ID NO: 62; SEQ ID NO: 69 and SEQ ID NO: 70; SEQ ID NO: 77 and SEQ ID NO: 78; SEQ ID NO: 85 and SEQ ID NO: 86; SEQ ID NO: 93 and SEQ ID NO: 94; or SEQ ID NO: 101 and SEQ ID NO: 102, respectively H and V L .

11. The use according to claim 6, wherein the antibody or antigen-binding fragment comprises a V having the amino acid sequence shown in SEQ ID NO: 117, 118, 119, 123, 124 or 125 H ; and a V having the amino acid sequence shown in SEQ ID NO: 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140 or 141 L .

12. The use according to claim 11, wherein the antibody or antigen-binding fragment comprises a V having the amino acid sequence shown in SEQ ID NO: 118 H ; and a V having the amino acid sequence shown in SEQ ID NO: 140 L .

13. Use according to claim 9, 10, 11 or 12, wherein the antibody comprises a heavy chain (HC) constant domain comprising the amino acid sequence shown in SEQ ID NO: 9, 10, 11, 12 or 13.

14. Use according to claim 9, 10, 11 or 12, wherein the antibody comprises a light chain (LC) constant domain comprising the amino acid sequence shown in SEQ ID NO:

14.

15. Use according to claim 9, 10, 11 or 12, wherein the antibody comprises a heavy chain (HC), the heavy chain comprising the amino acid sequence of SEQ ID NO: 142, 143, 144, 148, 149, 150, 167, 168, 169, 170, 174, 175, 176, 177, 178, 182, 183, 184, 185, 186, 187, 191, 192 or 193.

16. Use according to claim 9, 10, 11, 12, 13, 14 or 15, wherein the antibody comprises a light chain (LC), the light chain (LC) comprising the amino acid sequence shown in SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165 or 166.

17. Use according to claim 9, wherein the antibody comprises a heavy chain (HC) containing the amino acid sequence shown in SEQ ID NO: 143 and a light chain (LC) containing the amino acid sequence shown in SEQ ID NO: 165, and variants thereof in which the HC lacks a C-terminal lysine residue or C-terminal glycine-lysine.

18. Use according to any one of claims 1-17, wherein the cancer is pancreatic cancer, melanoma, breast cancer, lung cancer, head and neck cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma or blood tissue cancer.

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