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

Abstract

Humanized, unconjugated monoclonal antibodies specific for immunoglobulin-like transcript 3 (ILT3), also known as leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4), are described.

Description

Background of the Invention

[0001] (1) Field of the Invention

[0002] The present invention provides unconjugated monoclonal antibodies specific for the inhibitory receptor immunoglobulin-like transcript 3 (ILT3) expressed on the surface of myeloid immune cells.

[0003] (2) Description of Related Technology

[0004] Immunoglobulin-like transcript 3 (ILT3), named 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 immune responses (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. This gene is a member of the leukocyte immunoglobulin-like receptor (LIR) family, which is found in a gene cluster at chromosomal region 19q13.4. The encoded protein belongs to the subfamily B class of LIR receptors, which contain two or four extracellular immunoglobulin domains, a transmembrane domain and two to four ITIMs.

[0005] ILT3 is selectively expressed by myeloid antigen presenting cells (APC), 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, with a predicted molecular weight of about 47kD. The amino terminal portion of ILT3 begins with a 23 amino acid hydrophobic signal peptide, followed by an extracellular domain comprising two C2-type immunoglobulin superfamily domains, with an amino acid sequence as shown in SEQ ID NO:1, minus the C-terminal His tag. (The extracellular domain of macaque ILT3 has an amino acid sequence as 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, characterized by the presence of a motif spaced apart by 26 amino acid residues, reminiscent of the ITIM motif (natural-killer cell Ig receptor) determined in KIR as a binding site for protein tyrosine phosphatase SHP-1. ILT3 is expressed on immune cells, where it binds to the MHC class I molecules on antigen presenting cells and transduces negative signals that inhibit the stimulation of the immune response. This receptor can also play a role in antigen capture and presentation. ILT3 is considered to control inflammatory response and cytotoxicity to help focus the immune response and limit autoreactivity. Multiple transcript variants encoding different subtypes of ILT3 have been identified.

[0006] Patent publications disclosing the use of antibodies to modulate ILT3 activity and for inhibiting transplant rejection or for use in cancer or infectious disease treatment include U.S. 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 lymphocyte adhesion molecule (ALCAM), which is a ligand for ILT3, and in some embodiments provides a method of treating cancer comprising an antibody to CD166 or ALCAM. Also of interest are US Patent Nos. 7,777,008 and 8,901,281, which disclose monoclonal antibody 9B11 for use in a variety of therapeutics where it is desirable to upregulate the immune system for anti-cancer therapy and downregulate the immune system for suppression of transplant rejection.

[0007] Although patent publications 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-reactive 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 in therapeutic applications contraindicated. Therefore, there is a need for antibodies and antigen-binding fragments that specifically bind to ILT3 and have no measurable promiscuity to other related receptors. Summary of the invention

[0008] The present invention provides monoclonal antibodies and antigen-binding fragments that specifically bind to immunoglobulin-like transcript 3 (ILT3) without 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 a specific embodiment, the antibodies and antigen-binding fragments specifically bind to both human ILT3 and macaque ILT3. These antibodies and antigen-binding fragments are able to antagonize 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 have a high affinity for humanized NSG1 cells. TM In vivo studies in a mouse model system (The Jackson Laboratory, Bar Harbor, ME) showed that these antibodies may have the ability to reduce tumor burden and shift the cellular phenotype to a more activated state.

[0009] In samples from clinical trials, ILT3 expression, such as PD-L1, LAG-3 and GEP tags, 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 monotherapy or in combination with anti-PD-1 and / or anti-PD-L1 antibodies to enhance the responsiveness to anti-PD-1 or anti-PD-L1 antibodies, particularly in cancer treatments where cancer is unresponsive to anti-PD-1 or anti-PD-L1 monotherapy. In specific embodiments, the present invention provides chimeric or humanized anti-ILT3 antibodies. In certain embodiments, the antibody may be a fully human antibody that competes with the antibodies disclosed herein for binding to the ILT3 epitope disclosed herein.

[0010] The present invention provides an antibody or antigen-binding fragment comprising a heavy chain variable V having heavy chain complementarity determining regions (HC-CDRs) 1, 2 and 3. HOne, two or three complementarity determining regions (CDRs) of the light chain variable domain, and a light chain variable domain V having LC-CDR1, 2 and 3 L one, two or three CDRs of the present invention, wherein the antibody or antigen-binding fragment can specifically bind to human ILT3, wherein the binding of the antibody or antigen-binding fragment can be determined by cell ELISA or Biacore.

[0011] 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 described 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 described 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 described 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.

[0012] The present invention further provides an antibody or antigen-binding fragment that binds to human ILT3, comprising a heavy chain (HC), wherein the heavy chain variable domain (V H ) comprises a heavy chain complementary 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 an amino acid sequence having 3, 2 or 1 differences with an amino acid sequence selected from SEQ ID NO: 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 specific 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 complementary determining region (HC-CDR) 3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 23, 49, 57, 65, 73, 81, 89, 97 and 105, or an amino acid sequence having 3, 2 or 1 differences with an amino acid sequence selected from the group consisting of SEQ ID NO: 23, 49, 57, 65, 73, 81, 89, 97 and 105. In a specific embodiment, the amino acid sequence differences are conservative changes / substitutions.

[0013] 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 from one or more of the amino acid sequences described 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 described 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 described 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.

[0014] The present invention further provides an antibody or antigen-binding fragment that binds to human ILT3, comprising (a) HC having a variable domain (V H ), comprising a variable domain complementarity determining region (HC-CDR) 1 having an amino acid sequence as shown in SEQ ID NO: 17, 47, 55, 63, 71, 79, 87, 95 or 103; a HC-CDR2 having an amino acid sequence as shown in SEQ ID NO: 18, 48, 56, 64, 72, 80, 88, 96 or 104; a HC-CDR3 having an amino acid sequence as shown 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 a combination thereof; and (b) a light chain (LC) having a variable domain (V L ), comprising a variable domain complementarity determining region (LC-CDR) 1 having an amino acid sequence as set forth in SEQ ID NO: 27, 50, 58, 66, 74, 82, 90, 98 or 106; a LC-CDR2 having an amino acid sequence as set forth in SEQ ID NO: 43, 51, 59, 67, 75, 83, 91, 99 or 107; a LC-CDR3 having an amino acid sequence as 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 a combination thereof. In a specific embodiment, the amino acid sequence differences are conservative changes / substitutions.

[0015] In another embodiment of the antibody or antigen-binding fragment, HC-CDR1 has the amino acid sequence of SEQ ID NO: 17; HC-CDR2 has the amino acid sequence of SEQ ID NO: 19, 20 or 21; HC-CDR3 has the amino acid sequence of SEQ ID NO: 23; and LC-CDR1 has the amino acid sequence of SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, 41 or 42; LC-CDR2 has the amino acid sequence of SEQ ID NO: 43; and LC-CDR3 has the amino acid sequence of SEQ ID NO: 44; and variants thereof wherein one or more of the HC-CDR and LC-CDR has 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 of SEQ ID NO: 17; HC-CDR2 has the amino acid sequence of SEQ ID NO: 20; HC-CDR3 has the amino acid sequence of SEQ ID NO: 23; and LC-CDR1 has the amino acid sequence of SEQ ID NO: 41; LC-CDR2 has the amino acid sequence of SEQ ID NO: 43; and LC-CDR3 has the amino acid sequence of SEQ ID NO: 44; and variants thereof wherein one or more of the HC-CDR and LC-CDR has 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.

[0017] In another embodiment of the antibody or antigen-binding fragment, the antibody or antigen-binding fragment comprises (a) V H , which has a member selected from human V H 1. V H 2. V H 3. V H 4. V H 5. and V H 6 family, 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 member selected from human V κ 1. V κ 2. V κ 3. V κ 4. V κ 5. V κ 6. Vλ 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. 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 a specific embodiment, the amino acid sequence differences are conservative changes / substitutions.

[0018] 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 a combination 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 a combination thereof. In a specific embodiment, the amino acid sequence differences are conservative changes / substitutions.

[0019] 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 a combination thereof compared to the amino acid sequence of a native IgG1, IgG2, IgG3 or IgG4 isotype HC constant domain. In a specific aspect, the constant domain may comprise a C-terminal lysine or may lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide.

[0020] In a specific embodiment, the heavy chain constant domain is of human IgG1 isotype, which has been modified to have reduced or minimal effector function. In a further aspect, the minimal effector function is due to an effector-less Fc mutation, which may comprise or consist of a mutation N297A or D265A / N297A as determined using Kabat numbering, in which case the minimal effector function is due to aglycosylation (see, e.g., the amino acid sequence shown in SEQ ID NO: 211, wherein the N297A mutation corresponds to amino acid position 180; the D265A mutation (if present) would correspond to amino acid position 148). In a specific aspect, the IgG1 has been modified to comprise or consist of L234A, L235A and D265S mutations as determined using Kabat numbering to render the Fc effector lower (see, e.g., the amino acid sequence set forth in SEQ ID NO: 12 or 13, wherein the L234A, L235A and D265S mutations correspond to amino acid positions 117, 118 and 148, respectively).

[0021] In another aspect, the HC constant domain is of the human IgG4 isotype and further comprises a proline substitution for the serine residue at position 228 (EU numbering), which corresponds to position 108 of SEQ ID NO: 9 or 10 (serine at position 108).

[0022] 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 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or a combination thereof compared to the amino acid sequence of a native human κ or λ LC constant domain. In a specific embodiment, the amino acid sequence difference is a conservative change / substitution.

[0023] In another embodiment of the antibody or antigen-binding fragment, the antibody comprises (i) V H , which has a member selected from human V H 1. V H 2. V H 3. V H 4. V H 5 and V H 6 family and a human IgG1 or IgG4 HC constant domain, or a variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or a combination thereof compared to the amino acid sequence of a native human IgG1 or IgG4 isotype HC constant domain; and (ii) V L , which has a member selected from human V κ 1. Vκ 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 family framework and human kappa or lambda LC constant domain or a variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or a combination thereof compared to the amino acid sequence of a native human kappa or lambda LC constant domain. In a specific embodiment, the amino acid sequence difference is a conservative change / substitution.

[0024] In another embodiment of the antibody or antigen-binding fragment, the antibody comprises (i) a human V H 2 Family Framework V H and have people V κ 5 V of the Family Framework L ; (ii) a human IgG1 or IgG4 HC constant domain or a variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or a combination thereof compared to the amino acid sequence of a native human IgG1 or IgG4 isotype HC constant domain; and (iii) a human κ LC constant domain or a variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or a combination thereof compared to the amino acid sequence of a native human κ LC constant domain. In a specific embodiment, 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 human V H 1 Family Framework V H and have people V κ 5 People in the Family Framework V L; (ii) human IgG4 HC constant domain or a variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or a combination thereof compared to the amino acid sequence of a native human IgG4 isotype HC constant domain; and (iii) human κ LC constant domain or a variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or a combination thereof compared to the amino acid sequence of a native human κ LC constant domain. In a specific embodiment, the amino acid sequence differences are conservative changes / substitutions.

[0026] In another embodiment of the antibody or antigen-binding fragment, the antibody or antigen-binding fragment comprises a V sequence having an amino acid sequence as 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. H and V L .

[0027] In another embodiment of the antibody or antigen-binding fragment, the antibody or antigen-binding fragment comprises a V having an amino acid sequence as shown in SEQ ID NO: 117, 118, 119, 123, 124 or 125. H and a V having an amino acid sequence as shown in SEQ ID NO: 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140 or 141. L .

[0028] In another embodiment of the antibody or antigen-binding fragment, the antibody or antigen-binding fragment comprises a V having an amino acid sequence as shown in SEQ ID NO: 118. H and V having the amino acid sequence shown in SEQ ID NO: 140 L .

[0029] In another embodiment of the antibody, the antibody comprises a HC constant domain comprising the amino acid sequence shown in SEQ ID NO: 9, 10, 11, 12 or 13. In a specific aspect, the HC constant domain comprising the amino acid sequence shown in SEQ ID NO: 9, 11, 12 or 13 may lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide. In a specific embodiment, the HC constant domain comprises the amino acid sequence shown in SEQ ID NO: 10.

[0030] In another embodiment of the antibody, the antibody comprises an LC constant domain comprising the amino acid sequence shown in SEQ ID NO:14.

[0031] In another embodiment of the antibody, the antibody comprises a HC comprising an 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 a specific aspect, the HC comprising the amino acid sequence of 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 a specific embodiment, the HC comprises an amino acid sequence as shown in SEQ ID NO: 143 or 177. In a specific embodiment, the HC further lacks a C-terminal glycine as shown in SEQ ID NO: 177.

[0032] In another embodiment of the antibody, the antibody comprises a LC comprising the amino acid sequence of SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165 or 166. In a specific embodiment, the LC comprises the amino acid sequence of SEQ ID NO: 165.

[0033] In another embodiment of the antibody, the antibody comprises a HC having an amino acid sequence as shown in SEQ ID NO: 143 and a LC comprising an amino acid sequence as shown in SEQ ID NO: 165. In a specific aspect, the HC comprising the amino acid sequence as shown in SEQ ID NO: 143 lacks a C-terminal lysine residue or a C-terminal glycine-lysine dipeptide.

[0034] 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 sequence shown 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, wherein the epitope comprises the amino acid sequence shown 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.

[0035] The present invention further provides a chimeric, humanized or recombinant human antibody or antigen-binding fragment that binds to ILT3, wherein the binding cross-blocking antibody or the antibody that competes for binding comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 15 and a light chain having an amino acid sequence as 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 an amino acid sequence as shown in SEQ ID NO: 3, 4, 5, 6, 7 and 8, wherein the antibody comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 15 and a light chain having an amino acid sequence as shown in SEQ ID NO: 16.

[0036] 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.

[0037] 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.

[0038] 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, stomach cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral cavity or pharynx cancer, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or blood tissue cancer.

[0039] The present invention also provides a method for treating cancer in a subject, comprising administering to the subject simultaneously or continuously 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. In one embodiment, the antagonist of PD-1 is an antibody or 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 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.

[0040] 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.

[0041] The present invention further provides an antibody or antigen-binding fragment disclosed or claimed herein for use in treating cancer in a subject.

[0042] 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, stomach 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 cavity or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or a cancer of hematologic tissue.

[0043] The present invention further provides an antibody or antigen-binding fragment disclosed or claimed herein for use in 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.

[0044] In one embodiment, the PD-1 antagonist 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.

[0045] In one embodiment, the PD-L1 or PD-L2 antagonist 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.

[0046] In another embodiment, the anti-PD-1 antibody is nivolumab, pembrolizumab, cemiplimab, or pidilizumab, and the PD-L1 inhibitor is dulumab, atezolizumab, avastin, YW243.55.S70, MPDL3280A, MEDI-4736, MSB-0010718C, or MDX-1105.

[0047] The present invention further provides the use of the antibodies or antigen-binding fragments disclosed or claimed herein for treating cancer.

[0048] 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.

[0049] 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, stomach 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 cavity or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or a cancer of hematologic tissue.

[0050] 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 specific embodiment, the composition comprises a mixture of an antibody comprising a heavy chain having a C-terminal lysine and an antibody comprising a heavy chain lacking a C-terminal lysine. In a specific embodiment, the composition comprises an antibody disclosed herein, wherein the main antibody form comprises a heavy chain having a C-terminal lysine. In a specific embodiment, the composition comprises an antibody disclosed herein, wherein the main antibody form comprises a heavy chain lacking a C-terminal lysine. In a specific embodiment, the composition comprises an antibody disclosed herein, wherein about 100% of the antibodies in the composition comprise a heavy chain lacking a C-terminal lysine. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1A , Figure 1B , Figure 1C , Figure 1D , Figure 1E and Figure 1FA comparison of the selectivity of various of the anti-ILT3 antibodies disclosed herein for monoclonal antibody 9B11 and mouse IgG1 (mIgG1) using a cell-based ELISA format is shown. Figure 1A ), macaque ILT3( Figure 1B ), human ILT5( Figure 1C ), human ILT7( Figure 1D ), human ILT8 ( Figure 1E ), or human ILT11( Figure 1F ) were tested in CHO-K1 cells with monoclonal antibodies p40B5 (LB179.40B5.1A1), p49C6 (LB181.49C6.1A1), and p52B8 (lb181.52B8.1B1); antibody 9B11 (U.S. Pat. No. 7,777,008 having the amino acid sequence of SEQ ID NO:33 (light chain) and SEQ ID NO:34 (heavy chain)) and mouse IgG1.

[0052] Figure 2A Data features are shown for binding affinity, isoelectric point, monomeric species purity, and thermal stability measurements for variants of mAb 10. Terminology: "huILT3" refers to human ILT3; "rhILT3" refers to macaque 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.

[0053] Figure 2B mAb 10 (M64V V H 1 SEC purity of humanized light chain variants of IgG4) versus melting temperature. L 1-V L 8 refers to variants having the amino acid sequences shown in SEQ ID NOs: 126-133, respectively.

[0054] Figure 3A Shown by chimeric anti-ILT3 52B8 mouse 52B8 V H Parent / Human IgG4 (S228P): Mouse 52B8 Parent V LDeuterium-labeled differential heatmap of amino acid residues of the extracellular domain of human ILT3 bound by a human κ antibody ("c58B2"; mAb 73). These hexapeptide domains, which contain the epitope 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 extracellular domain of ILT3. The amino acid sequence of the human extracellular domain with a C-terminal His tag is shown in SEQ ID NO: 1.

[0055] Figure 3B The first and second views of the surface structure model of the extracellular domain of human ILT3 are shown. The dark area of ​​the model shows the location of the hexapeptide domain containing the human ILT3-His epitope bound by c58B8 (mAb 73).

[0056] Figure 3C is a ribbon diagram showing the location of epitopes on the extracellular domain of ILT3: 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).

[0057] Figure 3D Deuterium labeling heatmap showing amino acid residues of the extracellular domain of human ILT3 bound by antibody ZM4.1.

[0058] Figure 3E Deuterium labeling differential heatmap showing amino acid residues of the extracellular domain of human ILT3 bound by antibody DX446.

[0059] Figure 3F Deuterium labeling heatmap showing amino acid residues of the extracellular domain of human ILT3 bound by antibody DX439.

[0060] Figure 3G Deuterium labeling heatmap showing amino acid residues of the extracellular domain of human ILT3 bound by antibody 9B11.

[0061] Figure 4Free c52B8 (mAb 73) concentrations in the blood after multiple doses in humanized tumor models (Panc08.13 and SK-MEL-5) are shown. Free c52B8 concentrations are represented by circles and squares. The dotted line represents simulated historical antibody levels after IV bolus administration of 1, 3, 10, or 30 mg / kg of humanized IgG4 in C57BL / 6J mice.

[0062] Figure 5A Human dendritic cell (DC) functional assays are shown, showing anti-ILT3 chimeric antibodies in which the V from p52B8 was fused to IgG4 Fc (c52B8; mAb 73), IgG1 Fc (mAb 78), or IgG1(N297A)Fc (mAb 76). H and V L Has comparable ability to activate dendritic cells (DC). Human immature DCs were prepared and differentiated into CD11c+ dendritic cells with GM-CSF (1000U / mL) and IL-4 (1000U / mL) within 5 days. These cells were treated with IL-10, LPS (Gram-negative bacterial cell wall component and TLR4 ligand (Raetz et al.Ann.Rev.Biochem.71:635–700(2002)), and different concentrations of the indicated antibodies for 42 hours. The data shown are the average and sd of two technical replicates. This experiment represents four independent studies. Control IgG had no effect (not shown).

[0063] Figure 5B and Figure 5C Humanized 52B8 (lot 26AVY; mAb 46) was shown to be indistinguishable from c52B8 (mAb 73) in a human DC functional assay using DCs from two different healthy human donors. The data shown are the mean and sd of two technical replicates. The data shown are representative of three independent studies using these two donors.

[0064] Fig. 6A and Figure 6B Shown that anti-ILT3 antibody c52B8 (mAb 73) and humanized anti-ILT3 antibody 52B8 (mAb46; lot 26AVY) reduced the inhibitory capacity of bone marrow-derived suppressor cells (MDSC). The T cell inhibition assay was performed with a 4:1 T cell to MDSC ratio. The data shown are the mean and sd of three technical replicates at the level of the T cell assay step. The experimental representation shown was two independent studies using PBMC from the same two donors, with qualitatively similar results.

[0065] Figure 7 c52B8 was shown to inhibit the growth of SK-MEL-5 tumors in SK-MEL-5 human-NSG mice carrying SK-MEL-5 subcutaneous tumors. Animals were randomly assigned to treatment based on tumor volume on day 21 after transplantation, and 20 mg / kg of c52B8 or isotype control was given subcutaneously once a week starting on day 21. The data shown in the top graph are mean values ​​and std. errors (nine per group). Individual animal tumor growth curves are shown in the middle and bottom graphs. Body weights dropped to similar degrees in both the control and 52B8 groups. This study represents three independent studies.

[0066] Fig. 8A , Figure 8B , Figure 8C and Fig.8D The effect of c52B8 on tumor growth and immune activation in the SK-MEL-5hu-NSG model is shown. Fig. 8A Tumor growth curves are shown. Figure 8B CyTOF quantification of TILs collected 7 days after the second dose is shown: CD4 + Regulatory T cells and CD4 + CD69 expression levels on T cells; Figure 8C sHLA-G levels in plasma harvested at the end of the study are shown; Fig.8D Demonstration of human CD3 in tumors + IHC analysis of T cell infiltration, 4 tumors in each group.

[0067] Fig. 9A , Fig. 9B , Fig. 9C and Fig.9D Shown is the effect of c52B8 and pembrolizumab combination in Panc 08.13 human-NSG mice. Fig. 9A Tumor growth curves are shown. Fig. 9B CyTOF quantification showing % regulatory T cells and CD69 expression levels on CD4+ T cells from tumors harvested at the end of the study; Fig. 9C Plasma sHLA-G levels in endpoint blood samples are shown; Fig.9D Shown are plasma IFNγ and IL-8 levels in endpoint blood samples quantified using 10plex MSD (Meso Scale Discovery).

[0068] Fig.10It was shown that in an MDSC / T cell inhibition assay, at a 4:1 T cell to MDSC ratio, the humanized anti-ILT3 antibody 52B8 (mAb 46) reduced the suppressive capacity of MDSCs to a comparable extent to the chimeric anti-ILT3 antibody c52B8 (mAb 73).

[0069] Fig.11 Shown is the effect of the combination of humanized anti-ILT3 antibody 52B8 (mAb 46) and pembrolizumab in an MDSC / T cell inhibition assay at a T cell to MDSC ratio of 4:1 or 8:1 using MDSC cells obtained from human donor D001003835.

[0070] Fig.12 Shown is the effect of the combination of humanized anti-ILT3 antibody 52B8 (mAb 46) and pembrolizumab in an MDSC / T cell inhibition assay at a T cell to MDSC ratio of 8:1 using MDSC cells obtained from human donor D001003180.

[0071] Fig.13 Shown is the effect of the combination of humanized anti-ILT3 antibody 52B8 (mAb 46) and pembrolizumab in an MDSC / T cell inhibition assay at a 4:1 T cell to MDSC ratio using MDSC cells obtained from human donor D001003507.

[0072] Fig.14 Shown is the effect of the combination of humanized anti-ILT3 antibody 52B8 (mAb 46) and pembrolizumab in an MDSC / T cell inhibition assay at a T cell to MDSC ratio of 8:1 using MDSC cells obtained from human donor D001003428.

[0073] Fig.15 Shown is the effect of the combination of humanized anti-ILT3 antibody 52B8 (mAb46) and pembrolizumab in a mixed lymphocyte reaction of IL-10-polarized human monocyte-derived dendritic cells with allogeneic CD8+ T cells, with interferon gamma (IFNγ) measured in the culture supernatant after four days of incubation as a readout of T cell activation. DETAILED DESCRIPTION

[0074] The present invention provides unconjugated monoclonal antibodies specific for the inhibitory receptor immunoglobulin-like transcript 3 (ILT3) expressed on the surface of myeloid immune cells.

[0075] definition

[0076] "Immunoglobulin-like transcript 3" (abbreviated herein as "ILT3", and also referred to as LIR-5, LILRB4, or CD85k), as used herein and unless otherwise indicated, refers to a 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 D3).

[0077] As used herein, "antibody" refers to a complete immunoglobulin, including recombinantly produced forms, and includes any form of an antibody that exhibits a desired biological activity. Therefore, it is used in the broadest sense, specifically including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, bispecific antibodies, humanized camelid heavy chain antibodies, and non-human / human chimeric antibodies. "Parent antibody" is an antibody obtained by exposing the immune system to an antigen before the antibody is modified for its intended use, such as humanization of a non-human antibody used as a human therapeutic antibody.

[0078] In one embodiment, an "antibody" refers to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) 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 some naturally occurring IgG, IgD and IgA antibodies, the heavy chain constant region consists of three domains, CH1, CH2 and CH3. In some naturally occurring antibodies, each light chain consists of a light chain variable region or domain (abbreviated here as V L ) and a light chain constant region or domain. The light chain constant region consists of one domain, CL. 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 people V L The 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, V λ 3. V λ 4. V λ 5. Vλ 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.

[0079] V H and V L The V regions are further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L It consists of three CDR regions and four FR regions, which are arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The assignment of amino acids to each domain is usually based on the following definition: Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5 th ed.; NIHPubl. 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.

[0080] Typically, although antibodies contain six CDRs, V H Up three, V LThe above three, but the prior art recognizes that, in most cases, the CDR3 region of the heavy chain is the main determinant of antibody specificity, and examples of specific antibody generation based solely on 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 region of antibodies according to sequence); see also Chothia and Lesk (1987) J. Mol. Biol. 196:901-917 (defining the CDR regions of antibodies in terms of structure)).

[0081] The following general rules, shown in Table 1, can be used to identify CDRs in antibody sequences. There are rare instances where these nearly invariant features do not occur; however, the Cys residue is the most conserved feature.

[0082]

[0083]

[0084] Typically, the basic antibody structural unit comprises a tetramer. Each tetramer includes two pairs of identical polypeptide chains, each pair having a "light" chain (about 25 kDa) and a "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 primarily responsible for antigen recognition. The carboxyl terminal portion of the heavy chain can define a constant region, which is primarily responsible for the effector function of the antibody. Typically, human light chains are divided into κ and λ light chains. In addition, human heavy chains are typically 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 chain and the heavy chain, the variable region and the constant region are connected 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, NY (1989).

[0085] The heavy chain of the antibody may or may not include a terminal lysine (K) residue, or a terminal glycine and lysine (GK) residue. Therefore, in a specific embodiment of the anti-ILT3 antibody herein, it includes a lack of a terminal lysine, but the heavy chain constant region amino acid sequence shown herein terminated with a glycine residue, further including an embodiment in which a terminal glycine residue is also lacking. This is because the terminal lysine and sometimes glycine and lysine can be cut together during antibody expression, or cut off when introduced into the human body, without obvious adverse effects on antibody efficacy, stability or immunogenicity. In some cases, the nucleic acid molecule encoding the heavy chain can intentionally omit a codon encoding a terminal lysine or a codon encoding a terminal lysine and a glycine.

[0086] 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 a 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.

[0087] As used herein, a "Fab fragment" comprises a light chain and a 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 cleavage of an antibody.

[0088] As used herein, a "Fab' fragment" comprises a light chain and a portion or fragment of a heavy chain, which comprises VH The CH1 domain and the region between the CH1 and CH2 domains allow for the formation of interchain disulfide bonds between the two heavy chains of the two Fab' fragments to form a F(ab')2 molecule.

[0089] As used herein, a "F(ab')2 fragment" comprises two light chains and two heavy chains, wherein the two light chains and the two heavy chains comprise V H The F(ab')2 fragment is composed of two Fab' fragments, which are bound together by the disulfide bonds between the two heavy chains. The "F(ab')2 fragment" can be a product of pepsin cleavage of an antibody.

[0090] As used herein, an "Fv region" includes the variable regions from both the heavy and light chains, but lacks the constant regions.

[0091] 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 fragments are screened for utility in the same manner as intact antibodies. Antigen binding portions can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.

[0092] As used herein, the "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

[0093] As used herein, "diabodies" refer to small antibody fragments with two antigen-binding sites, which fragments contain a light chain variable domain (V L ) connected to the heavy chain variable domain (V H -V L or V L -V H). By using a linker that is too short to allow pairing between two domains on the same chain, these domains are forced to pair with the complementary domains of another chain and create two antigen binding sites. Diabodies are described more fully in, for example, 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.

[0094] As used herein, a "bispecific antibody" is an artificial hybrid antibody having two different heavy / light chain pairs and therefore two different binding sites. For example, a bispecific antibody may comprise a first heavy chain / light chain pair comprising a heavy chain and a light chain of a first antibody, which comprises at least six CDRs of an anti-ILT3 antibody disclosed herein or an embodiment in which one or more of the six CDRs have one, two or three amino acid substitutions, additions, deletions or a combination thereof, and a second heavy / light chain pair comprising a heavy chain and a light chain of a second antibody specific for an antigen of interest other than ILT3. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or connection 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 "diabodies" (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).

[0095] As used herein, an "isolated" antibody or antigen-binding fragment thereof is at least partially free of other biomolecules from the cells or cell cultures 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 the host cells or their growth medium. In general, the term "isolated" is not intended to refer to the complete absence of such biomolecules or the absence of water, buffers or salts, nor to the components of a pharmaceutical preparation comprising the antibody or fragment.

[0096] As used herein, "monoclonal antibody" refers to a substantially homogeneous antibody population, that is, the antibody molecules constituting the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in small amounts. In contrast, conventional (polyclonal) antibody preparations typically include a plurality of different antibodies having different amino acid sequences that are typically specific for different epitopes in their variable domains. The modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous antibody population, and is not to be interpreted as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies used 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 techniques such as those 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.

[0097] As used herein, a "chimeric antibody" is an antibody having a variable domain from a first antibody and a constant domain from a second antibody, wherein (i) the first and second antibodies are from different species (US 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., a variable domain from an IgG1 antibody and a constant domain from an IgG4 antibody. In one aspect, the variable domain is obtained from a non-human antibody, e.g., a mouse antibody ("parent antibody"), and the constant domain sequence is obtained from a human antibody. In another aspect, the variable domain is a humanized variable domain from a mouse antibody and a constant domain from a human antibody.

[0098] As used herein, "humanized antibody" refers to an antibody form comprising sequences from human and non-human (e.g., mouse, rat) antibodies. Typically, a humanized antibody will comprise all of at least one and usually two variable domains, wherein the hypervariable loops correspond to those of non-human immunoglobulins, and all or substantially all of the framework (FR) regions are sequences of those human immunoglobulin sequences. A humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region (Fc).

[0099] "Humanization" (also known as "remodeling" or "CDR transplantation") is now 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). Engineered mAbs are engineered using molecular biology techniques, but simple CDR transplantation of rodent complementarity determining regions (CDRs) into human frameworks often results in loss of binding affinity and / or specificity of the original mAb. In order to humanize antibodies, the design of humanized antibodies includes variants, such as conservative amino acid substitutions in CDR residues, and reverse substitutions (reverse mutations) from rodent mAb residues to human framework regions. Positions can be identified or recognized by structural analysis by sequence comparison or by analyzing homology models of the 3D structure of the variable region. The process of affinity maturation has recently used phage libraries to change the amino acids at selected positions. Similarly, many methods have been used to select the most suitable human framework for transplanting rodent CDRs. As the data set of known parameters of antibody structure increases, the improvement and refinement of these techniques also increase. Consensus 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 only the surface residues of the rodent sequence with the most common residues found in human monoclonal antibodies, an approach known as "resurfacing" or "veneer." Human or humanized antibodies are generally substantially non-immunogenic in humans.

[0100] As used herein, "non-human amino acid sequence" refers to an amino acid sequence with respect to an antibody or immunoglobulin that is characteristic of an amino acid sequence of a non-human mammal. The term does not include an amino acid sequence of an antibody or immunoglobulin obtained from a fully human antibody library, wherein the diversity in the library is generated by computer (see, e.g., U.S. Pat. Nos. 8,877,688 or 8,691,730).

[0101] As used herein, "effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the 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 receptor); and B cell activation.

[0102] As used herein, "conservatively modified variants" or "conservative substitutions" refer to amino acids that are replaced by other amino acids with similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), so that changes can often be made without changing the biological activity of the protein. Those skilled in the art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially change the biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.)). In addition, substitutions of amino acids that are similar in structure or function are less likely to destroy biological activity. Exemplary conservative substitutions are set forth in Table 2.

[0103]

[0104] As used herein, the term "epitope" or "antigenic determinant" refers to a site on an antigen (e.g., ILT3) to which an immunoglobulin or antibody specifically binds. Epitopes within protein antigens can be formed either by continuous amino acids (usually linear epitopes) or by non-continuous amino acids juxtaposed by tertiary folding of the protein (usually conformational epitopes). Epitopes formed by continuous amino acids are usually, but not always, retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are typically lost when treated with denaturing solvents. Continuous linear epitopes are included in a peptide domain on the antigen that includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. A non-continuous conformational epitope comprises one or more peptide domains or regions on an antigen bound by an antibody, interspersed with one or more amino acids or peptide domains that are not bound by the antibody, each domain independently comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids. Methods for determining which epitopes a given antibody binds to (i.e., epitope mapping) are well known in the art, including, for example, immunoblotting and immunoprecipitation assays, in which overlapping or continuous peptides (e.g., from ILT3) are tested for reactivity with a given antibody (e.g., anti-ILT3 antibody). Methods for determining the spatial conformation of an epitope include techniques in the art and described herein, such as X-ray crystallography, two-dimensional nuclear magnetic resonance, and HDX-MS (e.g., see Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996)).

[0105] The term "epitope mapping" refers to the process of identifying molecular determinants on an antigen that are 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).

[0106] The term "binding to the same epitope" relates to two or more antibodies and refers to a combination of amino acid residues or amino acid fragments that the antibodies bind to the same segment, as determined by a given method. Techniques for determining whether an antibody binds to the "same epitope on ILT3" as the antibodies described herein include epitope mapping methods, such as X-ray analysis of antigen: antibody complex crystals, thereby providing atomic resolution of the epitope, and HDX-MS. Other methods for monitoring the binding of antibodies to antigen fragments (e.g., proteolytic fragments) or antigen mutation variations, in which the loss of binding is caused by modification of amino acid residues within the antigen sequence, are generally considered to be indicative of epitope composition (e.g., alanine scanning mutagenesis--Cunningham & Wells (1985) Science 244: 1081). In addition, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity separate specific short peptides from a combinatorial phage display peptide library.

[0107] An antibody that "competes with another antibody for binding to a target, such as ILT3" refers to an antibody that inhibits (partially or completely) the binding of another antibody to a target, i.e., ILT3. Known competition assays can be used to determine whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of another antibody to a target. In certain embodiments, an antibody competes with another antibody and inhibits the binding of the other antibody to a 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 first incubated with the target). 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, NY, USA 1999. Competing antibodies bind to the same epitope, an overlapping epitope, or a neighboring epitope (e.g., as demonstrated by steric hindrance).

[0108] 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)).

[0109] 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.

[0110] 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, for example, 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. For example, an antibody that specifically binds to human ILT3 may also cross-react with ILT3 from certain non-human primate species (e.g., macaque or cynomolgus monkey).

[0111] As used herein, "isolated nucleic acid molecule" refers to DNA or RNA of genomic, mRNA, cDNA or synthetic origin, or some combination thereof, which is not associated with all or part of a polynucleotide in which the isolated polynucleotide is found in nature. Or connected to a polynucleotide that is not essentially connected. For the purposes of this disclosure, it should be understood that a nucleic acid molecule "comprising" a specific nucleotide sequence does not include a complete chromosome. In addition to the specific sequence, an isolated nucleic acid molecule "comprising" a specific nucleic acid sequence may include up to ten or even up to twenty or more coding sequences for other proteins or parts or fragments thereof, or may include operably linked regulatory sequences that control the expression of the coding region of the described nucleic acid sequence, and / or may include vector sequences.

[0112] As used herein, "treatment" refers to the internal or external administration of a therapeutic agent, such as a composition containing any antibody or antigen-binding fragment thereof of the present invention, to a subject or patient suffering from one or more diseases. The agent has a symptom or suspected disease for which the agent has therapeutic activity or preventive activity. Generally, the drug is administered in an amount effective to alleviate one or more disease symptoms in the subject or population being treated, whether by inducing regression of such symptoms or inhibiting their development to any clinically measurable degree. The amount of therapeutic agent effective to alleviate any particular disease symptom may vary according to factors such as the disease state, the age and weight of the patient, and the ability of the drug to induce a desired response in the subject. Whether the disease symptoms are alleviated can be assessed by any clinical measurement commonly used by a physician or other skilled healthcare provider to assess the severity or progression of the symptom. The term further includes delaying the development of symptoms associated with the condition and / or alleviating the severity of the symptoms of the condition. The term also includes improving existing uncontrollable or unwanted symptoms, preventing other symptoms, and improving or preventing the root causes of such symptoms. Therefore, the term means that a beneficial result has been conferred to a human or animal subject suffering from a condition, disease or symptom or having the potential to develop such a condition, disease or symptom.

[0113] As used herein, "treatment" as it applies to human or veterinary subjects refers to therapeutic treatment as well as diagnostic applications. "Treatment" as applied to human or veterinary subjects encompasses contacting or exposing an antibody or antigen-binding fragment of the invention to a human or animal subject.

[0114] As used herein, "therapeutically effective amount" refers to the amount of a particular substance sufficient to achieve the desired effect in the 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.

[0115] 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 for PD-1, PD-L1 (B7-H1) and PD-L2 (B7-DC), have been identified and 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 known as an immunosuppressive protein that negatively regulates TCR signaling (Ishida, Y. et al. (1992) EMBO J. 11: 3887-3895; Blank, C. et al. (Epub 2011) December 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, lead to a decrease in tumor-infiltrating lymphocytes, a decrease in 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). 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 of PD-1 with PD-L2 is also blocked (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99: 12293-7; Brown et al. (2003 J. Immunol. 170: 1257-66).

[0116] Antibodies and antigen-binding fragments

[0117] 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. Anti-ILT3 antibodies increase the activity of antigen presenting cells and dendritic cells, reduce the activity of monocyte repressors, and increase T cell activation. Therefore, the present invention also includes the use of anti-ILT3 antibodies in monotherapy for the treatment of cancer and with anti-PD-1 or anti-PD-L1 antibodies for the treatment of cancer in the first, second or third line therapy.

[0118] Anti-ILT3 antibodies include any antibody disclosed herein according to the amino acid sequence, and include any antibody comprising (i) at least one, two, three, four, five, or six CDRs of an antibody disclosed herein according to the amino acid sequence; or (ii) although there is no CDR amino acid sequence disclosed herein, it binds to the same epitope on ILT3 as the antibody disclosed in the present invention, and can modulate ILT3 receptor signaling, so that the antibody increases the activity of antigen presenting cells and dendritic cells, reduces the activity of suppressing monocytes, and increases the activation ability of T cells. In specific aspects, the antibody has no measurable binding to human ILT5, human ILT7, human ILT8, and human ILT11, as determined in a cell ELISA or in a Biacore assay using 10 μg / mL antibody. The term specifically excludes antibodies comprising at least one CDR of antibody ZM4.1 or antibody 9B11 or U.S. Patent Nos. 7,777,008 and 8,901,281 or U.S. Patent Nos. 20090202544, 20150110714. 20150139986, and 20170267759; and any other antibodies disclosed in International Publications WO2013043569, WO2013181438, WO2014116846, WO2016049641, WO2016127427, WO2018089300 and WO2018148494.

[0119] Anti-ILT3 antigen-binding fragments and the like include any protein or peptide comprising a molecule comprising (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, it binds to the same epitope on ILT3 as the antibody disclosed in the present invention, and can modulate ILT3 receptor signals, such that the antigen-binding fragment increases the activity of antigen-presenting cells and dendritic cells, reduces the activity of suppressing monocytes, and increases the priming ability of T cells. In specific aspects, the antigen-binding fragment has no measurable binding to human ILT5, human ILT7, human ILT8, and human ILT11, as determined in a cell ELISA or in a Biacore assay using 10 μg / mL of the anti-ILT3 antigen-binding fragment. The term specifically excludes antigen-binding fragments comprising at least one CDR of antibody ZM4.1 or antibody 9B11 or U.S. Patent Nos. 7,777,008 and 8,901,281 or U.S. Patent Nos. 20090202544, 20150110714. 20150139986, and 20170267759; and any other antibodies disclosed in International Publications WO2013043569, WO2013181438, WO2014116846, WO2016049641, WO2016127427, WO2018089300 and WO2018148494.

[0120] In a further embodiment, the anti-ILT3 antibody includes any antibody that comprises (i) at least HC-CDR3 of an antibody disclosed herein according to an amino acid sequence; or (ii) although there is no HC-CDR3 amino acid sequence disclosed herein, it binds to the same epitope on ILT3 as the antibody disclosed in the present invention, and can modulate ILT3 receptor signaling, so that the antibody increases the activity of antigen presenting cells and dendritic cells, reduces the activity of suppressing monocytes, and increases the priming ability of T cells. In a specific aspect, the antibody has no measurable binding to human ILT5, human ILT7, human ILT8, and human ILT11, as determined in a cell ELISA or in a Biacore assay using 10 μg / mL antibody. The term specifically excludes antibodies 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 Nos. 20090202544, 20150110714. 20150139986, and 20170267759; and any other antibodies disclosed in International Publications WO2013043569, WO2013181438, WO2014116846, WO2016049641, WO2016127427, WO2018089300 and WO2018148494.

[0121] Anti-ILT3 antigen-binding fragments and the like include any protein or peptide comprising a molecule comprising (i) at least a portion of an anti-ILT3 antibody disclosed herein according to an amino acid sequence, (ii) at least HC-CDR3 of an antibody disclosed herein according to an amino acid sequence, or (iii) although not having the HC-CDR3 amino acid sequence disclosed herein, it 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 suppressing monocytes, and increases the priming ability of T cells. In specific aspects, the antigen-binding fragment has no measurable binding to human ILT5, human ILT7, human ILT8, and human ILT11, as determined in a cell ELISA or in a Biacore assay using 10 μg / mL of the anti-ILT3 antigen-binding fragment. The term specifically excludes antigen-binding fragments comprising at least one CDR of antibody ZM4.1 or antibody 9B11 or U.S. Patent Nos. 7,777,008 and 8,901,281 or U.S. Patent Nos. 20090202544, 20150110714. 20150139986, and 20170267759; and any other antibodies disclosed in International Publications WO2013043569, WO2013181438, WO2014116846, WO2016049641, WO2016127427, WO2018089300 and WO2018148494.

[0122] In a specific 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 comprising the HC-CDR3 of the anti-ILT3 antibody molecule disclosed herein or the H3-CDR3 as shown in Table 3.

[0123] In a specific 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 comprising HC-CDR1, HC-CDR2, HC-CDR3, LC-CDR1, LC-CDR2 and LC-CDR3 disclosed herein or in Table 3.

[0124]

[0125] 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 comprising a heavy chain variable domain (V H), which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 22, 49, 57, 65, 73, 81, 89, 97 and 105, or an amino acid sequence having 3, 2 or 1 differences with an amino acid sequence selected from the group consisting of 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 sequence shown in SEQ ID NO: 3, 4, 5, 6, 7 and 8. In a specific embodiment, the amino acid sequence difference is a conservative change / substitution.

[0126] In a specific embodiment, the anti-ILT3 antibody is a humanized or chimeric anti-ILT3 antibody disclosed herein. In a specific 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 an anti-ILT3 antibody disclosed herein or competes for binding with an anti-ILT3 antibody disclosed herein, and the antibody comprises less than three CDRs of an anti-ILT3 antibody disclosed herein or does not contain CDRs of an anti-ILT3 antibody disclosed herein.

[0127] The present invention further provides an antibody or an antigen-binding fragment thereof, which comprises (i) at least six complementary 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 thereof; wherein the six CDRs of the anti-ILT3 antibody comprise a heavy chain (HC)-CDR1 having an amino acid sequence as shown in SEQ ID NO: 17, 47, 55, 63, 71, 79, 87, 95 or 103; an HC-CDR2 having an amino acid sequence as shown in SEQ ID NO: 18, 48, 56, 64, 72, 80, 88, 96, or 104; an HC-CDR3 having an amino acid sequence as shown in SEQ ID NO: 22, 49, 57, 65, 73, 81, 89, 97 or 105; NO:27, 50, 58, 66, 74, 82, 90, 98 or 106; LC-CDR2 having an amino acid sequence as shown in SEQ ID NO:43, 51, 59, 67, 75, 83, 91, 99 or 107; LC-CDR3 having an amino acid sequence as 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 a specific embodiment, the amino acid sequence difference is a conservative change / substitution.

[0128] In a specific embodiment, the present invention provides an antibody or antigen-binding fragment comprising six CDRs of an anti-ILT3 antibody, wherein the anti-ILT3 antibody comprises a heavy chain (HC)-CDR1 having an amino acid sequence as shown in SEQ ID NO: 17; an HC-CDR2 having an amino acid sequence as shown in SEQ ID NO: 19, 20 or 21; an HC-CDR3 having an amino acid sequence as shown in SEQ ID NO: 23, 24, 25 or 26; a light chain (LC)-CDR1 having an amino acid sequence as shown in SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, 41 or 42; an LC-CDR2 having an amino acid sequence as shown in SEQ ID NO: 43; and an LC-CDR3 having an amino acid sequence as shown in SEQ ID NO: 44.

[0129] In a specific embodiment, the present invention provides an antibody or antigen-binding fragment comprising six CDRs of an anti-ILT3 antibody, wherein the anti-ILT3 antibody has a heavy chain (HC)-CDR1 having an amino acid sequence as shown in SEQ ID NO: 17; an HC-CDR2 having an amino acid sequence as shown in SEQ ID NO: 20; an HC-CDR3 having an amino acid sequence as shown in SEQ ID NO: 23; a light chain (LC)-CDR1 having an amino acid sequence as shown in SEQ ID NO: 41; an LC-CDR2 having an amino acid sequence as shown in SEQ ID NO: 43; and an LC-CDR3 having an amino acid sequence as shown in SEQ ID NO: 44.

[0130] In a specific embodiment, 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 H ), having a framework selected from the group consisting of: human V H 1. V H 2. V H 3. V H 4. V H 5 and V H 6 family, 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 ), having a framework selected from the group consisting of: 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 a combination thereof.

[0131] In a specific embodiment, 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, which comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or a combination thereof compared to the amino acid sequence of a native IgG1, IgG2, IgG3 or IgG4 isotype.

[0132] In a specific embodiment, the present invention provides the above-mentioned 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 a combination thereof compared to the amino acid sequence of a native human κ or λ light chain domain.

[0133] In a specific embodiment, the present invention provides the above-mentioned antibody or antigen-binding fragment, wherein the antibody comprises (i) a human heavy chain variable domain (V H ), which has a member selected from human V H 3 family framework, and the human light chain variable domain (V L ), which has a member selected from human V κ 1. V κ 3, and V κ 4 family; (ii) human IgG1 or IgG4 heavy chain (HC) constant domain or its variants comprising 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 a native IgG1 or IgG4 isotype; (iii) human κ or λ light chain constant domain or its variants comprising 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 a native human κ or λ light chain domain. In a specific embodiment, the amino acid sequence difference is a conservative change / substitution.

[0134] In a specific embodiment, 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 H ) and the light chain variable domain (V L): SEQ ID NO:15 and SEQ ID NO:16, respectively; 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, respectively; SEQ ID NO:93 and SEQ ID NO:94; SEQ ID NO:101 and SEQ ID NO:102.

[0135] In a specific embodiment, 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 H ) having an amino acid sequence as shown in SEQ ID NO: 117, 118, 119, 120, 121, 122, 123, 124 or 125. H ) and a light chain variable domain (V L domain) having an amino acid sequence as shown in SEQ ID NO: 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140 or 141. L ).

[0136] In a specific embodiment, 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 H ) and a light chain variable domain (V L ).

[0137] In a specific embodiment, 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 a variant of SEQ ID NO:9, 11, 12 or 13, wherein the HC lacks the C-terminal lysine or glycine lysine.

[0138] In a specific embodiment, 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.

[0139] In a specific embodiment, the present invention provides the above-mentioned antibody or antigen-binding fragment, wherein the antibody comprises a heavy chain (HC), wherein 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, and variants of 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 the C-terminal lysine or glycine-lysine.

[0140] In a specific embodiment, the present invention provides the above-mentioned antibody or antigen-binding fragment, 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.

[0141] In a specific embodiment, 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 of SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165 or 166, and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 143, 144, 148, 149, A variant of the HC having an amino acid sequence of 150, 167, 168, 169, 170, 174 or 175, wherein the HC lacks the C-terminal lysine or glycine lysine.

[0142] In a specific embodiment, the present invention provides an antibody selected from the group consisting of the antibodies shown in Table 4.

[0143] In a specific embodiment, 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 shown in SEQ ID NO: 143 and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO: 165, as well as variants in which HC lacks C-terminal lysine or glycine lysine.

[0144] In a specific embodiment, 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 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or a combination thereof compared to the amino acid sequence of a native IgG1, IgG2, IgG3 or IgG4 isotype, and a variant thereof in which the HC lacks a terminal lysine or a glycine lysine.

[0145] In some embodiments, different constant domains can be fused to a V domain comprising the CDRs provided herein. L and V H In certain embodiments, the V region comprising the CDRs provided herein may be H The region is fused to a human IgG1, IgG2, IgG3 or IgG4 heavy chain (HC) constant domain or variants thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions, or a combination thereof compared to the amino acid sequence of a native or wild-type IgG1, IgG2, IgG3 or IgG4 isotype, as well as variants in which the HC lacks a variant C-terminal lysine or a glycine lysine.

[0146] In certain embodiments, the anti-ILT3 antibody (or antigen-binding fragment) has altered effector function and may comprise a heavy chain constant domain other than native (wild-type) human IgG1, such as a human IgG1 with a mutation that eliminates or minimizes one or more effector functions, including the ability to bind complement, human IgG4 or hybrid human IgG1 / human IgG4, and variants in which the HC lacks a C-terminal lysine or glycine-lysine.

[0147] Although natural human IgG1 antibodies provide long half-life and effector functions, such as complement activation and antibody-dependent cellular cytotoxicity, such activity may not be desirable for all uses of the antibody. Therefore, in certain embodiments, it is desirable that the heavy chain constant domain or Fc has little or reduced effector function ("low effector"). In those cases, the anti-ILT3 HC variable domains can be fused to human IgG4 constant domains, which are generally known to be low effector, or to IgG1 constant domains that have been mutated to be low effector. Compared to polypeptides comprising the wild-type IgG Fc region, these molecules with lower effectors have minimal or reduced binding to human FcγRIIIA, FcγRIIA and Fcγ.RI, wherein the affinity to human FcγRIIIA, FcγRIIA and FcγRI is reduced by 1.15 to 100 times compared to polypeptides comprising wild-type IgG constant domains, wherein the antibody-dependent cell-mediated cytotoxicity (ADCC) induced by the molecules is 0-20% of the ADCC induced by polypeptides comprising wild-type human IgG1 type constant domains.

[0148] Therefore, in a specific embodiment, the present invention includes a chimeric or humanized anti-ILT3 antibody comprising a human IgG4 constant domain and an antigen-binding fragment thereof. In another embodiment, the human IgG4 constant domain can be modified to be different from the position corresponding to position 228 in the EU system of the natural (wild-type) human IgG4 constant domain (Swiss-Prot accession number P01861.1), and the 241st position in the Kabat system, wherein the natural serine at position 108 (Ser108) of the HC constant domain is replaced by proline (Pro), for example, see SEQ ID NO: 9. This modification prevents the formation of potential interchain disulfide bonds between the cysteine ​​(Cys106) at position 106 and the cysteine ​​(Cys109) at position 109, which corresponds to Cys226 and Cys229 in the EU system and Cys239 and Cys242 in the Kabat system, which may interfere with the formation of appropriate intrachain disulfide bonds. See Angal et al. Mol. Imunol. 30:105 (1993); see also (Schuurman et al., Mol. Immunol. 38:1-8, (2001); SEQ ID NOs:14 and 41). In a specific embodiment, the human IgG4 constant domain may include a L235E substitution in addition to the S228P substitution.

[0149] In another embodiment, the chimeric or humanized anti-ILT3 antibody can be fused to a modified human IgG1 constant domain that has been modified to be effector-low. In one embodiment, the human IgG1 HC can comprise replacement 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 a specific embodiment, the antibody comprises a human IgG1 heavy chain (HC) constant domain or a variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or a combination thereof compared to the amino acid sequence of a native IgG, which provides an antibody with reduced or minimal effector function. In a specific aspect, the IgG1 has been modified to comprise or consist of L234A, L235A and D265S mutations to render the Fc effector lower. Other mutations that can be used to render the IgG1 Fc effector lower can be found in U.S. Patent No. 8,969,526.

[0150] In another embodiment, a human IgG1 HC is modified to be N-glycosylated lacking an asparagine (Asn) residue near position 297 of the HC. The consensus sequence for N-glycosylation is Asn-Xaa-Ser / Thr (where Xaa is any amino acid except Pro); in IgG1, the consensus sequence for N-glycosylation 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 a codon for another amino acid, such as Gln. Alternatively, the codon for Ser can be replaced with a codon for Pro or the codon for threonine can be replaced with any codon except a codon for Ser, for example, N297A or N297D. Such modified IgG1 molecules have little or no detectable effector function. Alternatively, all three codons are modified.

[0151] In another embodiment, the human IgG1 constant domain is modified to include one or more amino acid substitutions selected from E233P, L234A, L235A, L235E, N297A, N297D, D265S and P331S, wherein the residues are numbered according to the EU index of Kabat, and wherein the polypeptide exhibits reduced affinity with human FcγRIIIa and / or to FcγRIIa and / or FcγRI compared to a polypeptide comprising a wild-type IgG constant domain region. In a specific embodiment, the human IgG constant domain comprises, for example, L234A, L235A and D265S substitutions as shown in SEQ ID NO: 4. In a specific embodiment, the human IgG1 constant domain comprises an amino acid substitution at position Pro329 and at least one other amino acid substitution E233P, L234A, L235A, L235E, N297A, N297D, D265S and P331S. These and other alternatives are disclosed in WO9428027; WO2004099249; WO20121300831, US Patent Nos. 9,708,406; 8,969,526; 9,296,815; Sondermann et al. Nature 406, 267-273 (20 Jul. 2000).

[0152] In one embodiment of the invention, the anti-ILT3 antibody or antigen-binding fragment includes an embodiment in which one or more of the six CDRs have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, including a full tetramer structure having two light chains and two heavy chains (including constant regions). The variable region of each light chain / heavy chain pair forms an antibody binding site. Therefore, generally, a complete antibody has two binding sites. Except for bispecific antibodies, the two binding sites are generally the same.

[0153] In a specific embodiment, the present invention provides the anti-ILT3 antibodies shown in Table 4. H The antibodies disclosed herein bind to human ILT3, except for those antibodies comprising a substitution of a tryptophan residue at position 101 of .

[0154]

[0155]

[0156]

[0157]

[0158]

[0159] 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 near the boundary between the D1 and D2 domains of the extracellular domain of ILT3. The epitope determined using HDX-MS shows 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 further embodiments, 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 is 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 specific embodiment, the epitope comprises or consists of a peptide domain shown in SEQ ID NO:3, 4, 5, 6, 7, and 8.

[0160] The present invention also provides chimeric, humanized or human antibodies or antigen-binding fragments 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 sequence shown in SEQ ID NO: 3, 4, 5, 6, 7, and 8 as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis.

[0161] In another embodiment, the present invention further provides a chimeric, humanized or human antibody or antigen-binding fragment that binds to an epitope on ILT3, wherein the epitope comprises or consists of amino acids within a peptide domain shown in one or more of SEQ ID NOs: 3, 4, 5, 6, 7, and 8. In certain embodiments, the epitope comprises or consists of as determined by HDX-MS and shown in Figure 3A The heat map identifies the composition of each peptide domain.

[0162] The present invention further provides a chimeric, humanized or human antibody or antigen-binding fragment that cross-blocks the antibody binding to an epitope on ILT3, the antibody comprising a heavy chain variable domain having an amino acid sequence as shown in SEQ ID NO: 15 and a light chain variable domain having an amino acid sequence as 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, the peptide domain comprising or consisting of an amino acid sequence as shown in the amino acid sequence as 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 an amino acid within a peptide domain as shown in one or more of SEQ ID NO: 3, 4, 5, 6, 7, and 8. In certain embodiments, the epitope comprises or consists of at least one amino acid in each of the peptide domains as determined by HDX-MS.

[0163] The present invention further provides bispecific antibodies and antigen-binding fragments, which comprise a first antibody or antigen-binding fragment that binds to ILT3 and a second antibody or antigen-binding fragment that binds to 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 the group consisting of SEQ ID NOs: 22, 49, 57, 65, 73, 81, 89, 97, and 105, or having an amino acid sequence having 3, 2 or 1 differences from an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 49, 57, 65, 73, 81, 89, 97, and 105, wherein the first antibody binds to an ILT3 epitope comprising amino acids within the sequence of SEQ ID NOs: 3, 4, 5, 6, 7, and 8, and the second antibody binds to a molecule other than ILT3, and methods of use thereof.

[0164] The present invention further provides bispecific antibodies and antigen-binding fragments, which comprise a first antibody or antigen-binding fragment that binds to ILT3 and a second antibody or antigen-binding fragment that binds to a molecule other than ILT3, wherein the first antibody or antigen-binding fragment comprises at least an anti-ILT3 antibody or six CDRs thereof wherein one or more of the CDRs have one, two, or three amino acid substitutions, additions, deletions, or a combination thereof, and wherein the first antibody binds to an ILT3 epitope comprising amino acids within the sequence of SEQ ID NOs: 3, 4, 5, 6, 7, and 8, and the second antibody binds to a molecule other than ILT3, and methods of use thereof.

[0165] The invention further provides biparatopic antibodies (antibodies that have binding specificity for different epitopes on the same antigen) having a first heavy chain / light chain pair of a first antibody comprising at least a HC-CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 49, 57, 65, 73, 81, 89, 97, and 105, or having an amino acid sequence that has 3, 2, or 1 differences from an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 49, 57, 65, 73, 81, 89, 97, and 105; wherein the first heavy chain / light chain pair binds an ILT3 epitope comprising amino acids within the sequence of SEQ ID NOs: 3, 4, 5, 6, 7, and 8, and the second antibody binds a molecule other than ILT3, the second heavy chain / light chain pair of the second antibody having a specificity for an anti-ILT3 epitope that is different from the epitope recognized by the first heavy / light chain pair.

[0166] The present invention further provides biparatopic antibodies (antibodies with binding specificity for different epitopes on the same antigen) having a first heavy chain / light chain pair of a first antibody comprising at least six CDRs of an anti-ILT3 antibody or embodiments thereof wherein one or more of the CDRs have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof, wherein the first antibody binds an ILT3 epitope comprising amino acids within the sequence of SEQ ID NOs: 3, 4, 5, 6, 7, and 8, wherein the first heavy chain / light chain pair binds an ILT3 epitope comprising amino acids within the sequence of SEQ ID NOs: 3, 4, 5, 6, 7, and 8, and the second antibody binds a molecule other than ILT3, the second heavy chain / light chain pair of the second antibody having a specificity for an anti-ILT3 epitope that is different from the epitope recognized by the first heavy / light chain pair.

[0167] Pharmaceutical compositions and administration

[0168] To prepare a pharmaceutical or sterile composition of an anti-ILT3 antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof is mixed with a pharmaceutically acceptable carrier or excipient. See, for example, Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984) and US Pharmacopeial Convention (USP) 12601 Twinbrook Parkway, Rockville, MD 20852-1790, USA, which is continuously updated online.

[0169] Formulations of therapeutic and diagnostic agents can be prepared by mixing with pharmaceutical carriers, excipients or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions or suspensions (see, for example, 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).

[0170] 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 for administering antibody molecules are known in the art and are described below. The appropriate dose of the molecule used will depend on the age and weight of the subject and the specific drug used. The dose and treatment regimen of the anti-ILT3 antibody or antigen-binding fragment can be determined by a technician. In certain embodiments, the anti-ILT3 antibody or antigen-binding fragment is administered by injection (e.g., subcutaneously or intravenously) at a dose 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 administered 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 every other week at a dosage of about 10 to 20 mg / kg.

[0171] The mode of administration can vary. Suitable routes of administration are preferably parenteral or subcutaneous. Other routes of administration may include oral, transmucosal, intradermal, direct intraventricular, intravenous, intranasal, inhalation, insufflation or intra-arterial.

[0172] In certain embodiments, the anti-ILT3 antibody or antigen-binding fragment thereof can be administered by an invasive route, such as by injection. In other embodiments of the invention, the anti-ILT3 antibody or antigen-binding fragment thereof or a pharmaceutical composition thereof can be administered intravenously, subcutaneously, intra-arterially or by inhalation, aerosol delivery. Administration by a non-invasive route (e.g., oral; e.g., in a pill, capsule, or tablet) is also within the scope of the invention.

[0173] The composition can be administered with medical devices known in the art.For example, the pharmaceutical composition of the present invention can be administered by injection with a hypodermic needle, including, for example, a prefilled syringe or an autoinjector.

[0174] The pharmaceutical compositions disclosed herein may also be administered with a needle-free subcutaneous injection device; preferably, a needle-free subcutaneous injection device may be used, such as the devices disclosed in U.S. Pat. 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.

[0175] The pharmaceutical compositions disclosed herein can 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 drugs at a controlled rate; U.S. Patent No. 4,447,233 discloses a drug infusion pump for delivering drugs 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 with multiple chambers. Many other such implants, delivery systems and modules are well known to those skilled in the art.

[0176] The administration 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 enough therapeutic antibody to achieve an improvement in the target disease state while minimizing undesirable side effects. Therefore, the amount of the delivered biologic depends in part on the specific therapeutic antibody and the severity of the condition being treated. There is guidance for selecting appropriate doses of therapeutic antibodies (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. (2002) New Engl. J. Med. 346:784-790). 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).

[0177] The dosage regimen is adjusted to provide the best desired response (e.g., therapeutic response). For example, a single bolus may be administered, several separate doses may be administered over time, or the dose may be proportionally reduced or increased depending on the urgency of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniform dosage. As used herein, a dosage unit form refers to a physically discrete unit suitable as a unit dose for a subject to be treated; for example, a unit dose. Each unit contains a predetermined amount of active compound that is calculated to produce the desired therapeutic effect together with the desired pharmaceutical carrier. The specifications of the dosage unit form described herein are directly determined by (a) the unique characteristics of the antibody or antibody binding fragment and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the formulation field. Such active molecules are used to treat the sensitivity of individuals. (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. al. (20003) Cancer Immunol. Immunother. 52:133-144).

[0178] Use of the anti-ILT3 antibodies or antigen-binding fragments disclosed herein

[0179] The anti-ILT3 antibodies and antigen-binding fragments disclosed herein that are not contaminated with related ILTs can be used to specifically detect human ILT3 (e.g., in biological samples such as serum or plasma) using conventional immunoassays such as enzymes, linked immunosorbent assays (ELISAs), radioimmunoassays (RIA) or tissue immunohistochemistry. The present invention therefore provides a method for detecting human ILT3, comprising an anti-ILT3 antibody or antigen-binding fragment in a biological sample contacted with the biological sample, and detecting any anti-ILT3 antibody or antigen-binding fragment bound to human ILT3 or unbound anti-ILT3 antibodies or antigen-binding fragments 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 the detection of bound or unbound anti-ILT3 antibodies or antigen-binding fragments disclosed herein. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; and the like. 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 suitable radioactive materials include 125I, 131I, 35S and 3H.

[0180] In addition to labeling the anti-ILT3 antibody or antigen-binding fragment, human ILT3 can be measured in biological fluids by competitive immunoassays 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, the biological sample, the labeled ILT3 standard, and the anti-ILT3 antibody or antigen-binding fragment are combined, and the amount of the 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 the anti-ILT3 antibody or antigen-binding fragment bound to the labeled standard ILT3.

[0181] The anti-ILT3 antibodies or antigen-binding fragments disclosed herein can also be used to detect ILT3 from species other than humans, particularly ILT3 from primates (eg, cynomolgus monkeys or macaques).

[0182] Methods for upregulating immune responses in vivo

[0183] The anti-ILT3 antibodies or antigen-binding fragments disclosed herein can be used as immunostimulatory compositions, for example, used alone or as part of a vaccine or combined therapy, to promote the activation of B cells and / or T cells, for example, whether Th1 cells or Th2 cells are activated. That is, the anti-ILT3 antibodies or antigen-binding fragments disclosed herein can be used as adjuvants used in combination with target antigens to enhance the immune response in vivo to the target antigen. For example, in order to stimulate the 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 in the same or different compositions simultaneously, or sequentially in time, to enhance the immune response). The target antigens and anti-ILT3 antibodies or antigen-binding fragments disclosed herein can be formulated together as a single pharmaceutical composition or a separate composition. In one embodiment, the target antigens and anti-ILT3 antibodies or antigen-binding fragments disclosed herein are administered to a subject simultaneously. Alternatively, in some cases, it may be desirable to administer the antigen first and then administer the anti-ILT3 antibody or antigen-binding fragment 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 a Th1 response, and then administer the anti-ILT3 antibody or antigen-binding fragment disclosed herein alone or with antigen enhancement to convert the immune response to a Th2 response). In a preferred embodiment, the anti-ILT3 antibody or antigen-binding fragment disclosed herein is administered at the time of priming with the antigen, i.e., at the time of the first antigen administration. For example, day -3, -2, -1, 0, +1, +2, +3. A particularly preferred day for administration of the anti-ILT3 antibody or antigen-binding fragment disclosed herein is day -1.

[0184] In one embodiment, the anti-ILT3 antibodies or antigen-binding fragments disclosed herein are administered together with an antigen of interest. The antigen of interest is an antigen to which an immune response is desired. For example, an antigen of interest is an antigen capable of stimulating immune protection in a subject against an attack by an infectious agent derived from the antigen. It is also contemplated that an anti-ILT3 antibody or antigen-binding fragment disclosed herein is administered to increase an immune response without administering an antigen.

[0185] Thus, exemplary antigens of interest include those derived from infectious agents, wherein an immune response to the antigen is used to prevent or treat a disease caused by the agent. Such antigens include, but are not limited to, viral, bacterial, fungal or parasitic proteins and 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.

[0186] Typical antigens of interest can be classified as follows: protein antigens, such as ceruloplasmin and serum albumin; and bacterial antigens, such as echinic acid, flagellar antigens, capsular polysaccharides, extracellular bacterial products and toxins; glycoproteins and glycolipids; viruses, such as animal, plant and bacterial viruses; combined and synthetic antigens, such as protein / hapten conjugates, are molecules preferentially expressed by tumors 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, which triggers a cellular immune response in the host. Non-limiting examples of viral antigens that can 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, HIV Env protein or its components gp120 and gp41, HIV Nef protein and 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 deletion in the mucin region of the molecule (Yang et al., NatMED 6: 886 (2000), smallpox antigen, hepatitis A, B or C virus, human rhinovirus such as type 2 or type 14, herpes simplex virus, poliovirus type 2 or 3, hand, foot and mouth disease virus (FMDV), rabies virus, rotavirus, influenza virus, coxsackie virus, human papillomavirus (HPV) (e.g., papillomavirus type 16), its E7 protein and fragments containing the E7 protein or its epitope; 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 bacteria are those of Bordetella pertussis origin (e.g., P69 protein and filamentous hemagglutinin (FHA) antigen), Vibrio cholerae, Bacillus anthracis, and antigens of Escherichia coli, such as Escherichia coli (E. coli) thermophilic toxin B subunit (LT-B), E. coli K88 antigen, and antigens of enterotoxigenic E. coli (E. coli). Other examples of antigens include Schistosoma mansoni P28 glutathione S-transferase antigen (P28 antigen) and flukes, mycoplasmas, ascaris, tapeworms, antigenic Chlamydia trachomatis and malarial parasites, for example, parasites of the genus Plasmodium or Babesia, such as Plasmodium falciparum, and peptides encoding immunogenic epitopes of the above antigens.

[0187] As used herein, the term "tumor-associated antigen" refers to an antigen that affects tumor growth or metastasis in a host organism. Tumor-associated antigens may be antigens expressed by tumor cells, or may be antigens expressed by non-tumor cells, but when expressed in this way, may promote the growth or metastasis of tumor cells. Types of tumor antigens and tumor-associated antigens include any known or hitherto unknown tumor antigens, including but not limited to bcr / abl antigens in leukemia, HPVE6 and E7 antigens of oncogenic viruses associated with cervical cancer, MAGE1 and MZ2-E antigens in or associated with melanoma, and MVC-1 and HER-2 antigens in or associated with breast cancer.

[0188] Infections, diseases or conditions 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 condition in which the host immune response plays a role in preventing infection, disease or condition. Disorders. Diseases, conditions 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 condition caused by or. Fungi, parasites, viruses or bacteria, diseases, conditions or infections caused by or associated with bioterrorism, listeriosis, Ebola, SARS, small pox, hepatitis A, hepatitis B, hepatitis C, human induced diseases and conditions caused by or associated with rotavirus, influenza, coxsackievirus, human papillomavirus, SIV, malaria, cancer (e.g., tumors) caused by or associated with rhinovirus, HIV and AIDS, herpes, polio, foot and mouth disease, rabies, diseases or disorders caused by or associated with Bordetella pertussis, Vibrio cholerae, Bacillus anthracis, Escherichia coli, flukes, mycoplasmas, arcus, tapeworms, Chlamydia trachomatis and Plasmodium, etc.

[0189] Immune response to tumor cells

[0190] Regulatory T cells play an important role in maintaining immunological self-tolerance by suppressing the immune response against autoimmune diseases and cancer. Therefore, in one embodiment, upregulating the immune response will be beneficial for enhancing the immune response in cancer. Therefore, the anti-ILT3 antibodies or antigen-binding fragments disclosed herein can be used to inhibit tumor growth or metastasis in the treatment of malignant tumors. The anti-ILT3 antibodies or antigen-binding fragments disclosed herein can be administered systemically or locally to the tumor site.

[0191] 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 resistance in the subject.

[0192] As used herein, the term "neoplastic disease" is characterized by malignant tumor growth or a disease state characterized by benign hyperproliferative and hyperplastic cells. The general medical meaning of the term "neoplasia" refers to "new cell growth" that results in a loss of responsiveness to normal growth controls (e.g., tumor cell growth).

[0193] As used herein, the terms "hyperproliferative", "hyperplastic" are used interchangeably and refer to an abnormal state or condition in those cells characterized by rapid proliferation or a tumor. The terms "malignant" and "neoplastic" are meant to include all types of hyperproliferative growth, hyperplastic growth, cancerous growth or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, and are related to the type of tissue pathology or invasion. "Hyperplasia" in the stage refers to cells that experience an abnormally high rate of growth. However, as used herein, the terms "neoplasia" and "hyperplasia" are used interchangeably, as revealed by their context, generally refer to cells that experience an abnormal cell growth rate, and neoplasms and hyperplasias include "tumors", which can be benign, pre-malignant or malignant.

[0194] The terms "neoplasia," "hyperplasia," and "tumor" are often used in conjunction with "cancer," which is an umbrella term for more than 100 diseases characterized by uncontrolled, abnormal growth of cells. Examples of cancer include, but are not limited to: breast cancer; colon; non-small cell lung, head and neck; colorectal lung; prostate; ovarian; renal melanoma; gastrointestinal (e.g., pancreatic and gastric) cancer; and osteogenic sarcoma.

[0195] 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, urinary 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, kidney cancer, testicular cancer, bile duct cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma and blood tissue cancer.

[0196] Immune response to infectious agents

[0197] The upregulation of the immune response can be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, in the case of viral infection, it may be useful to enhance the immune response by regulating ILT3. Since the anti-ILT3 antibodies or antigen-binding fragments disclosed herein can act to enhance the immune response, they will be useful in treatment in cases where faster or complete removal of pathogens such as bacteria and viruses would be beneficial.

[0198] As used herein, the term "viral infection" includes infection with organisms including, but not limited to, HIV (e.g., HIV-1 and HIV-2), human herpes virus, cytomegalovirus (particularly 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, paramyxovirus: respiratory syncytial virus, parainfluenza virus, measles virus, mumps virus, human papillomavirus (e.g., HPV6, 11, 16, 18, etc.), flavivirus (e.g., yellow fever virus, dengue virus, T-transmitted encephalitis virus, Japanese encephalitis virus) or influenza virus.

[0199] As used herein, the term "bacterial infection" includes infections with various bacterial organisms, including Gram-positive and Gram-negative bacteria. Examples include, but are not limited to, Neisseria, including Neisseria gonorrhoeae and Neisseria meningitidis, Streptococci, including Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus mutans; Haemophilus, including Haemophilus influenzae type B, non-typeable Haemophilus influenzae, Haemophilus ducreyi; Moraxella, including Moraxella catarrhalis, also known as Boula catarrhalis; Bordetella, including Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica; Mycobacterium, including Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium avium, Mycobacterium paratuberculosis, Mycobacterium smegmatis; Legionella species, including Lactobacillus pneumophila; Escherichia coli, including enterotoxigenic Escherichia coli, enterohemorrhagic Escherichia coli, enteropathogenic Escherichia coli; Vibrio cholerae, including Vibrio cholerae, Shigella, including S. sonnei, Streptococcus dysenteriae, Streptococcus flexneri; Yersinia, including Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Campylobacter jejuni, including Campylobacter jejuni and Escherichia coli. Salmonella, including Salmonella Typhi, Salmonella Paratyphi, Salmonella Cholera, Salmonella Enteritidis; Listeria, including Listeria monocytogenes; Helicobacter pylori, including Helicobacter pylori; Pseudomonas species, including Pseudomonas aeruginosa; Staphylococcus species, including Staphylococcus aureus, Staphylococcus epidermidis; Enterococci, including Enterococcus faecalis, Enterococcus faecalis; Clostridium, including Clostridium tetani, Clostridium botulinum, Clostridium difficile; Bacillus, including Bacillus anthracis; Corynebacterium, including Chlamydia diphtheriae; Borrelia burgdorferi (Borrelia spp.), including B.burgdorferi, B.garinii, B.afzelii, B.andersonii, B.hermsii; Ehrlichia, including E.equi and the pathogen of human granulocytic escherichia; Rickettsia, including R.rickettsii; Chlamydia, including Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci; Leptsiraspp., including L. interrogans; Treponema pallidum, 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.

[0200] 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 activation signal (e.g., antigen plus APC or polyclonal antibody) and reintroduced into the patient.

[0201] The anti-ILT3 antibodies or antigen-binding fragments disclosed herein can also be used prophylactically in vaccines against various pathogens. Immunity against pathogens such as viruses can be induced by vaccination with viral proteins and anti-ILT3 antibodies or antigen-binding fragments disclosed herein. Alternatively, expression vectors, i.e., encoding genes for antigens disclosed herein and anti-ILT3 antibodies or antigen-binding fragments, such as vaccinia virus expression vectors engineered to express viral proteins encoding nucleic acids and anti-ILT3 antibodies or antigen-binding fragments disclosed herein encoding nucleic acids, can be used for vaccination. Vaccines may be useful for pathogens including, for example, hepatitis B, hepatitis C, Epstein-Barr virus, cytomegalovirus, HIV-1, HIV-2, tuberculosis, malaria and schistosomiasis.

[0202] The present invention further includes an anti-ILT3 antibody or antigen-binding fragment disclosed herein conjugated to a diagnostic or therapeutic agent. The anti-ILT3 antibody or antigen-binding fragment disclosed herein can be used for diagnosis as part of a clinical testing program, such as monitoring the occurrence or progression of a tumor, for example, to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals using various positron emission tomography, and non-radioactive paramagnetic metal ions. The detectable substance can be coupled or bound directly or indirectly to the binding molecule through an intermediate (e.g., a linker known in the art) using techniques known in the art. U.S. Patent No. 4,741,900 discloses metal ions that can be bound to binding molecules. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; and the like. 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.

[0203] 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 cytocidal agent, a therapeutic agent or a radioactive metal ion, such as an α-emitter, such as, for example, 213Bi. Cytotoxins or cytotoxic agents include any substance that is harmful to cells. Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone dione, mitoxantrone, oryzalin, protestosterone, creatinine tetracaine, lidocaine, propranolol and puromycin and their analogs or homologs. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarboxyzine), alkylating agents (e.g., methoxyethylamine, thiopyradazole, melphalan, carmotine (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., dactinomycin), bleomycin, mithramycin and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine).

[0204] The present invention further relates to a therapy involving administration of an anti-ILT3 antibody or antigen-binding fragment disclosed herein to an animal, preferably a mammal, most preferably a human patient, to treat, detect and / or prevent one or more of the following: a disease, disorder or condition disclosed herein. The therapeutic compounds of the present invention include, but are not limited to, an anti-ILT3 antibody or antigen-binding fragment disclosed herein. The anti-ILT3 antibody or antigen-binding fragment 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.

[0205] 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, they are used to increase the number or activity of effector cells that interact with the binding molecules.

[0206] The anti-ILT3 antibodies or antigen-binding fragments disclosed herein can be administered alone or in combination with other types of treatments, such as immunostimulatory treatments or treatments intended to control the proliferation 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.

[0207] For therapeutic purposes, the 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 expressing ILT3, for veterinary purposes or as animal models of human disease, and the binding molecules cross-react with ILT3 (e.g., primates).

[0208] Combination

[0209] The anti-ILT3 antibody or antigen-binding fragment herein can be used in an uncoupled form or coupled to a second agent, such as a cytotoxic drug, a radioisotope or a protein such as a protein toxin or a viral protein. The method comprises administering an anti-ILT3 antibody or antigen-binding fragment herein alone or conjugated to a cytotoxic drug to a subject in need of such treatment. The anti-ILT3 antibody or antigen-binding fragment herein can be used to deliver a variety of therapeutic agents, such as cytotoxic moieties, such as therapeutic drugs, radioisotopes, plants, molecules or biological proteins (such as protein toxins) or particles (such as recombinant viral particles, such as through viral coat proteins) of fungal or bacterial origin, or mixtures thereof.

[0210] Other combination therapies

[0211] The anti-ILT3 antibodies or antigen-binding fragments disclosed herein can be used in combination with other therapies. For example, the combination therapy may include a composition 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 anticancer agents, cytotoxic or cytostatic agents, hormone therapy, vaccines and / or other immunotherapies. In other embodiments, the anti-ILT3 antibody or antigen-binding fragment thereof is administered with other therapeutic treatments, including surgery, radiation, cryosurgery, and / or hyperthermia. Such a combination therapy can advantageously utilize a lower dose of the administered therapeutic agent, thereby avoiding possible toxicity or complications associated with various monotherapies.

[0212] "In combination with..." is not meant to imply that the treatment and / or therapeutic agents must be administered simultaneously and / or formulated for delivery together, although these delivery methods are within the scope described herein. The anti-ILT3 antibody or antigen-binding fragment thereof may be administered simultaneously with or before or after one or more other additional therapies or therapeutic agents. The anti-ILT3 antibody or antigen-binding fragment thereof and other agents or treatment regimens may be administered in any order. Typically, each agent will be administered at a dose and / or schedule determined for that agent. It will also be recognized that the additional therapeutic agents used in the combination may be administered together in a single composition or separately in different compositions. Typically, it is desirable to use additional therapeutic agents in combination at a level that does not exceed that of using them alone. In some embodiments, the level of combined use will be lower than the level of use alone.

[0213] 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, an antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide. In some embodiments, the anti-PD-1 antibody is selected from nivolumab (OPDIVO, Bristol Myers Squibb, New York, New York), pembrolizumab (KEYTRUDA, Merck Sharp & Dohme Corp, Kenilworth, NJUSA), cetiplimab (Regeneron, Tarrytown), NY) or pidilizumab (CT-011). In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an 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 dulumab (IMFINZI, Astrazeneca, Wilmington, DE), atezolizumab (TECENTRIQ, Roche, Zurich, CH), or avastin (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.

[0214] MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in WO2007 / 005874. Antibody YW243.55.S70 is an anti-PD-L1 antibody described in WO2010 / 077634 (the heavy chain and light chain variable region sequences are shown in SEQ ID NOs: 20 and 21, respectively).

[0215] 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.

[0216] Pembrolizumab, also known as KEYTRUDA, Ambrolizumab, MK-3475 or SCH-900475, is a humanized anti-PD-1 antibody described in U.S. Pat. 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 chain and light chain of pembrolizumab are represented by the amino acid sequences shown in SEQ ID Nos: 225 and 226, respectively.

[0217] 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, for example, the anti-PD-1 antibodies disclosed in U.S. Patent 8609089; U.S. Publication No. 2010028330; and U.S. Publication No. 20120114649.

[0218] AMP-224 (B7-DCIg; Amplimmune; disclosed, for example, in WO2010 / 027827 and WO2011 / 066342) is a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD-1 and B7-H1.

[0219] 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. Pat. No. 7,943,743 and U.S. Publication No. 20120039906.

[0220] Other anti-PD-L1 binders include YW243.55.S70 (the heavy and light chain variable regions are shown as SEQ ID NOs: 20 and 21 in WO2010 / 077634) and MDX-1105 (also known as BMS-936559). It and other anti-PD-L1 binders are disclosed in WO2007 / 005874.

[0221] Reagent test kit

[0222] Also provided are kits comprising one or more components, including, but not limited to, an anti-ILT3 antibody or antigen-binding fragment thereof as 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 as a pure composition or in combination with a pharmaceutically acceptable carrier in a pharmaceutical composition.

[0223] In one embodiment, the kit comprises an 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 bottle).

[0224] In another embodiment, the kit comprises the combination anti-ILT3 antibodies or antigen-binding fragments thereof or pharmaceutical compositions thereof formulated together, optionally, in a pharmaceutical composition, in a single, common container.

[0225] If the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit may include a device for performing such administration. For example, the kit may include one or more hypodermic needles or other injection devices as described above. Thus, the present invention includes a kit comprising an injection device and an anti-ILT3 antibody or antigen-binding fragment thereof, for example, wherein the injection device comprises the antibody or fragment, or wherein the antibody or fragment is in a separate container.

[0226] The kit may include a package insert that includes information about the pharmaceutical compositions and dosage forms in the kit. Typically, such information helps patients and physicians to effectively and safely use the enclosed pharmaceutical compositions and dosage forms. For example, the following information about the combinations of the invention may be provided in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdose, appropriate dosage and administration, appropriate storage conditions, references, manufacturer / distributor information, and patent information.

[0227] Method for preparing antibodies and antigen-binding fragments thereof

[0228] Anti-ILT3 antibodies or antigen-binding fragments thereof disclosed herein can also be recombinantly produced. In this embodiment, the nucleic acid molecule encoding the antibody molecule can be inserted into a vector (plasmid or virus) and transfected or transformed into a host cell, where the nucleic acid molecule can be expressed and secreted from the host cell. Several methods for producing recombinant antibodies are known in the art.

[0229] In a specific aspect, the present invention provides nucleic acid molecules encoding HC and LC, wherein the HC comprises at least HC-CDR3 of an anti-ILT3 antibody disclosed herein or an embodiment in which the HC-CDR3 has one, two, or three amino acid substitutions, additions, deletions, or a combination 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 a combination thereof.

[0230] In a specific aspect, the present invention provides a nucleic acid molecule encoding HC and LC, wherein the HC comprises HC-CDR1, 2 and 3 of an anti-ILT3 antibody disclosed herein or an embodiment in which one or more of HC-CDR1, 2 and 3 have one, two or three amino acid substitutions, additions, deletions or a combination thereof, and wherein the LC comprises LC-CDR1, 2 and 3 of an anti-ILT3 antibody disclosed herein or an embodiment in which one or more of HC-CDR1, 2 and 3 have one, two or three amino acid substitutions, additions, deletions or a combination 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 a combination thereof.

[0231] In a specific aspect, the present invention provides a first expression vector comprising a nucleic acid molecule encoding a HC, wherein the HC comprises at least the HC CDR of an anti-ILT3 antibody disclosed herein or an embodiment in which one or more of the three HC CDRs have one, two or three amino acid substitutions, additions, deletions or a combination 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 a combination thereof, and a second expression vector comprising a nucleic acid molecule encoding a LC, wherein the LC comprises at least the LC CDR of an anti-ILT3 antibody disclosed herein or an embodiment in which one or more of the three LC CDRs have one, two or three amino acid substitutions, additions, deletions or a combination 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 a combination thereof.

[0232] In a specific aspect, the present invention provides a H and V L A nucleic acid molecule, wherein V H An embodiment comprising at least HC-CDR3 of an anti-ILT3 antibody disclosed herein, or an embodiment wherein the HC-CDR3 has one, two or three amino acid substitutions, additions, deletions or a combination thereof. In a further embodiment, V H and / or V L The variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or a combination thereof.

[0233] In a specific aspect, the present invention provides a H and V L A nucleic acid molecule, wherein the HC 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 have one, two or three amino acid substitutions, additions, deletions or combinations thereof, and wherein the V L An embodiment comprising LC-CDR1, 2 and 3 of an anti-ILT3 antibody disclosed herein or wherein one or more of HC-CDR1, 2 and 3 have one, two or three amino acid substitutions, additions, deletions or a combination thereof. In a further embodiment, V H and / or V L The variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or a combination thereof.

[0234] In a specific aspect, the present invention provides a coding V H A nucleic acid molecule, wherein the VH comprises the HC CDR of the anti-ILT3 antibody disclosed herein or an embodiment in which one or more of the three HC CDRs have one, two, or three amino acid substitutions, additions, deletions or a combination thereof, and / or wherein V H and / or V L The variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or a combination thereof, and encodes V L The nucleic acid molecule, the V L An embodiment comprising an LC CDR of an anti-ILT3 antibody disclosed herein or wherein one or more of the three LC CDRs thereof have one, two or three amino acid substitutions, additions, deletions or a combination thereof, and / or wherein 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 a combination thereof.

[0235] 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 Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., 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 (e.g., Trichoderma reesei) and yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris). In particular aspects, the host cell can be a prokaryotic host cell, such as Escherichia coli.

[0236] When a recombinant expression vector comprising a nucleic acid molecule encoding a heavy chain or 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 the antibody to be expressed in the host cell, or more preferably, the antibody is secreted into the culture medium in which the host cell is grown. The antibody can be recovered from the culture medium and further purified or processed to produce an antibody of the invention.

[0237] In a specific aspect, a host cell is transfected with an expression vector comprising a nucleic acid molecule encoding a HC and a LC, wherein the HC comprises an HC-CDR3 of an anti-ILT3 antibody disclosed herein or an embodiment thereof wherein the HC-CDR3 has one, two or three amino acid substitutions, additions, deletions or a combination 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 a combination thereof.

[0238] In a specific aspect, the host cell is transfected with an expression vector comprising a nucleic acid molecule encoding a HC and a LC, wherein the HC comprises HC-CDR1, 2 and 3 of an anti-ILT3 antibody disclosed herein or an embodiment in which one or more of the HC-CDR1, 2 and 3 have one, two or three amino acid substitutions, additions, deletions or a combination thereof, and wherein the LC comprises LC-CDR1, 2 and 3 of an anti-ILT3 antibody disclosed herein or an embodiment in which one or more of the HC-CDR1, 2 and 3 have one, two or three amino acid substitutions, additions, deletions or a combination 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 a combination thereof.

[0239] In a specific aspect, a host cell is transfected with a first expression vector and a second expression vector, wherein the first expression vector comprises a nucleic acid molecule encoding a HC, wherein the HC comprises the HC CDR of an anti-ILT3 antibody disclosed herein or an embodiment in which one or more of the three HC CDRs have one, two, or three amino acid substitutions, additions, deletions, or a combination 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 a combination thereof, and the second expression vector comprises a nucleic acid molecule encoding a LC, wherein the LC comprises the LC CDR of an anti-ILT3 antibody disclosed herein or an embodiment in which one or more of the three LC CDRs have one, two or three amino acid substitutions, additions, deletions or a combination 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 a combination thereof.

[0240] In a specific aspect, a host cell is transfected with an expression vector comprising a vector encoding V H and V L A nucleic acid molecule, wherein V H At least comprising the HC-CDR3 of the anti-ILT3 antibody disclosed herein or an embodiment thereof, wherein the HC-CDR3 has 1, 2 or 3 amino acid substitutions, additions, deletions or a combination thereof. In a further embodiment, V H and / or V L The variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or a combination thereof.

[0241] In a specific aspect, a host cell is transfected with an expression vector comprising a nucleic acid molecule encoding V H and V L , where VH The invention also includes HC-CDR1, 2 and 3 of the anti-ILT3 antibodies disclosed herein, or an embodiment in which one or more of HC-CDR1, 2 and 3 have one, two or three amino acid substitutions, additions, deletions or a combination thereof, and wherein V L An embodiment comprising LC-CDR 1, 2 and 3 of an anti-ILT3 antibody disclosed herein or wherein one or more of LC-CDR 1, 2 and 3 have 1, 2 or 3 amino acid substitutions, additions, deletions or a combination thereof. In a further embodiment, V H and / or V L The variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or a combination thereof.

[0242] In a specific aspect, a host cell is transfected with a first expression vector and a second expression vector, wherein the first expression vector comprises a nucleic acid molecule encoding V H , the V H Embodiments comprising at least the HC CDR of the anti-ILT3 antibody disclosed herein or one or more of its three HC CDRs have one, two or three amino acid substitutions, additions, deletions or a combination thereof, and / or wherein V H The variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or a combination thereof, and the second expression vector comprises a nucleic acid molecule encoding V L , the V L Embodiments disclosed herein wherein at least the LC CDR of the anti-ILT3 antibodies or one or more of the three LC CDRs have one, two or three amino acid substitutions, additions, deletions or combinations thereof, and / or wherein V L The variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions, or a combination thereof.

[0243] In a specific embodiment, HC and LC or V H and V L Expressed as a fusion protein in which the N-termini of 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).

[0244] The present invention further provides a plasmid or viral vector comprising a nucleic acid molecule encoding an 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 an anti-ILT3 antibody or antigen-binding fragment thereof disclosed herein, or one or more of its three CDRs having one, two or three amino acid substitutions, additions, deletions or a combination 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 a combination thereof, and an LC encoding an anti-ILT3 antibody or antigen-binding fragment thereof disclosed herein, or one or more of its three CDRs having one, two or three amino acid substitutions, additions, deletions or a combination 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 a combination thereof.

[0245] 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.

[0246] The present invention also provides a host cell comprising a plasmid or a viral vector comprising a nucleic acid molecule encoding the HC of 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 a combination 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 a combination thereof, and a plasmid or a viral vector comprising a nucleic acid molecule encoding the LC of 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 a combination 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 a combination thereof. In specific embodiments, the host cell is a CHO or HEK-293 host cell.

[0247] 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. The present invention further provides a plasmid or viral vector comprising a V encoding an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein. H or an anti-ILT3 antibody or an antigen-binding fragment thereof, wherein one or more of the three CDRs have one, two or three amino acid substitutions, additions, deletions or a combination thereof, and / or wherein V H The framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions, or a combination thereof, and encodes the V sequences of the anti-ILT3 antibodies or antigen-binding fragments thereof disclosed herein. L or an anti-ILT3 antibody or an antigen-binding fragment thereof, wherein one or more of the three CDRs have one, two or three amino acid substitutions, additions, deletions or a combination thereof, and / or wherein V L The framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions, or a combination thereof.

[0248] The present invention further provides a V encoding an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein. H A plasmid or viral vector containing a nucleic acid molecule or antigen-binding fragment thereof and a V encoding an anti-ILT3 antibody or antigen-binding fragment thereof disclosed herein L A plasmid or viral vector containing a nucleic acid molecule or antigen-binding fragment.

[0249] The present invention also provides a host cell comprising a plasmid or a viral vector comprising a nucleic acid molecule encoding a V or V fragment of an anti-ILT3 antibody or an antigen-binding fragment thereof disclosed herein. H 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 a combination thereof, and / or the V H The variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions, or a combination thereof, and a plasmid or viral vector comprising a nucleic acid molecule encoding a V region of an anti-ILT3 antibody or antigen-binding fragment thereof disclosed herein. L or an embodiment of an anti-ILT3 antibody or antigen-binding fragment thereof disclosed herein, wherein one or more of the three CDRs has one, two, or three amino acid substitutions, additions, deletions, or a combination thereof, and / or wherein V LThe variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions, or a combination thereof. In a specific embodiment, the host cell is a CHO or HEK-293 host cell.

[0250] Anti-ILT3 antibodies or their antigen-binding fragments can be recovered from culture medium using standard protein purification methods. In addition, a variety of known techniques can be used to enhance the expression of antibodies of the present invention (or other parts thereof) from production cell lines. For example, the glutamine synthetase gene expression system (GS system) is a common method for enhancing expression under certain conditions.

[0251] 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, e.g., Croset et al., J. Biotechnol. 161: 336-348. (2012)). Thus, the specific glycosylation pattern of an antibody will depend on the specific 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 independently of the glycosylation pattern that the antibody may have.

[0252] The following examples are intended to facilitate a further understanding of the present invention.

[0253] General approach

[0254] 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, describing 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).

[0255] 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 modifications, post-translational modifications, 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) Bio Directory, Piscataway, NJ, pp. 384-391). The 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).

[0256] 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; US Pat. No. 6,329,511).

[0257] An alternative approach to humanization is to 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).

[0258] The antibody can be coupled to, for example, a small drug molecule, an enzyme, a liposome, polyethylene glycol (PEG). The antibody can be used for treatment, diagnosis, kits or other purposes, and includes, for example, antibodies coupled to dyes, radioisotopes, enzymes or metals (e.g., 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).

[0259] Flow cytometry methods, including fluorescence activated cell sorting (FACS), can be used (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 use, e.g., as diagnostic reagents, 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).

[0260] Standard methods for immunohistology 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).

[0261] Software packages and databases are provided for determining, for example, antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments (see, for example, 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).

[0262] 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 No. 186005225, 1.7 micron, 200 angstroms, 4.6 mm x 150 mm) from Waters (Milford, MA). The column temperature was 25°C and 10 μL of sample was injected at 1 mg / mm 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. BEH200 SEC Protein Standard Mix (Part No. 186006518) purchased from Waters (Milford, MA) was used and injected at a dose of 10 ug to measure USP Resolution, theoretical plates, and tailing.

[0263] NANO-DSF (trade name modified differential scanning fluorescence method to determine the stability of proteins, using intrinsic tryptophan or tyrosine fluorescence): The temperature midpoint Tm of the thermal unfolding curve and the midpoint Tagg of the thermal polymerization curve are determined by NANO-DSF using a Prometheus NT.48 differential scanning fluorimeter (Nanotemper Technologies) controlled by PRTherm Control v2.0.4 software. The excitation power is 40% and the temperature is increased from 20°C to 95°C at a rate of 1°C / min. Tm and Tagg are measured automatically. The sample is prepared by diluting to 1 mg / mL in 20mM sodium acetate pH5.5 buffer and aspirating it into a Prometheus glass capillary (PR-L002) by capillary action.

[0264] Capillary isoelectric focusing (cIEF): cIEF was performed on an iCE3 system from Protein Simple (San Jose, CA) using iCE CFR software 4.1.1 for instrument control and data analysis. The cIEF cartridge used was Fc-coated (Protein Simple, 101701) and prepared according to the manufacturer's instructions. 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 of each 5.85 and 9.22 pI marker (Protein Simple) was prepared. The sample was injected for 60 seconds. Isoelectric focusing parameters were 1500 V for 1 min and 3000 V for 8 min. The pI was measured automatically using an internal pI marker as a two-point calibration standard. The calibrated data were converted to Empower format for further analysis and quantification and analyzed using Empower3.

[0265] Example 1

[0266] Hybridoma clone 52B8 is identified by standard mouse and rat immunization and hybridoma selection.Usually, in standard four-week footpad immunization, Balb / C mice or rats are immunized with human ILT3-HIS recombinant protein to produce hyperimmune response.Immortalized hybridomas are produced by the electrofusion of the main body lymphocytes from the draining lymph nodes and the P3 myeloma fusion partner.Hybridoma supernatant screens human CHO-human ILT3 cells in the ELISA binding assay based on primary cells.CHO parents, CHO-ILT3 SNP, CHO-rhesus ILT3, CHO-ILT5, CHO-ILT8 and CHO-ILT11 cells (see Example 2) are screened in the cell-based ELISA format.Subclones are carried out by limiting dilution on ILT3 specific and macaque positive hybridoma cells.Subclones are expanded to produce purified proteins so that additional tests for Biacore analysis and functional screening can be carried out. Table 5 shows 10 hybridoma clones that produced antibodies that were grouped together and had high affinity for human ILT3 as shown by CELISA and Biacore performed as disclosed in Examples 2 and 4, respectively.

[0267]

[0268]

[0269] Table 6 shows the amino acid sequences of the heavy and light chain variable domains of mAbs obtained from the above clones.

[0270]

[0271] To ultimately guide lead antibody selection, the antibodies were further analyzed and re-evaluated in a battery of biofunctional, 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.

[0272] Example 2

[0273] Selectivity of various anti-ILT3 antibodies

[0274] Cell-based ELISA (cELISA) was used to show the selectivity of various parent anti-ILT3 antibodies shown in Table 5 and humanized anti-ILT3 monoclonal antibody 9B11 (disclosed in U.S. Pat. No. 7,777,008, which has the amino acid sequence of SEQ ID NO: 33 (light chain) and SEQ ID NO: 34 (heavy chain)).

[0275] Mouse anti-human ILT3 antibodies were tested for binding to human ILT3 and cross-reactivity with macaque ILT3, human ILT5, human ILT7, human ILT8, and human ILT11 expressing CHO-K1 cells 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). Two days prior to the assay, 2 × 10 4 cells / well or 4×10 4Cells / well were plated. 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 / 05% Tween-20 using the cell washing program on the Biotek EL405x Select CW plate washer. Binding was detected using a HRP-conjugated goat anti-mouse IgG (Southern Biotech cat#1031-05) secondary antibody added at a dilution of 1:2000 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 stopped with TMB stop solution (KPL catalog number 50-85-06). The absorbance at 450nm-620nm was measured. Mouse IgG1 (MIgG1) was used as a control.

[0276] The results are shown in Figure 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 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 to macaque ILT3. The p52B8 clone 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 macaque ILT3.

[0277] Example 3

[0278] The parental mouse 52B8 heavy chain (V H ) and light chain (V L ) The variable domain sequences were compared to human germline sequences. Human framework sequences were selected that were closely homologous to the framework of the mouse antibody.

[0279] Mouse anti-human ILT3 mAb 52B8 cloned mouse V H The domain scored high for human heavy chain germline 3-07 in subgroup III and JH4 in the J region. Based on structural considerations, two framework substitutions (R87K and A97G) were introduced to maintain equivalent binding to the parent antibody. The mouse V L The domain scored high for human light chain germline 1-02 in kappa subgroup I. The mouse 52B8 CDRs were engineered onto the variable light chain sequences of 1-02 and JK2 in the J region. Based on structural considerations, three framework substitutions (M4L, S64A, and G72R) were introduced.

[0280] To generate humanized variants, humanized V H The sequence was cloned into a vector encoding the human IgG4 S228P heavy chain constant domain, and the humanized V L The domain was cloned into a vector encoding the constant domain of the kappa light chain. A total of two humanized V H (V H 1 and V H 2) and 8 personalized V L Computer sequence and structural analysis of mouse 52B8 revealed six potential "hot spots" on the molecule: V H -CDR2(M64) and V H - Two potential oxidation sites in CDR3 (W101), V H - A potential isomerization site in CDR2 (D62), 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.

[0281] Figure 2A Tables are provided showing data features for binding affinity, isoelectric point, monomeric species purity and thermal stability measurements for the designed humanized variants. Biacore was used to measure binding affinity, cIEF was used to measure pI, purity was determined by SE-UPLC, Tm and Tgg were determined by Nano-DSF. Figure 2B The SEC purity of various humanized light chain variants is shown as a function of melting temperature. The data are plotted as the values ​​obtained for each of the eight humanized light chain variants, indicating that V L 5 has both the highest purity and thermal stability. Figure 2A and Figure 2B Data, V L 5 was selected for the light chain.

[0282] Humanized V produced in transient CHO cells H 1 M64V / V L 5 A preliminary study was conducted. In unformulated humanized 52B8 V H 1 M64V / V L 5. The forced deamidation conditions of 50℃ incubation and high pH stress showed that V L-Deamidation of LC N34 in CDR1 (4.0% and 7.2%, respectively), and oxidation of W101 in HC-CDR3 was 15.4% at 1× light stress exposure. Substitution of N34 for Q34 maintained binding affinity to human and macaque ILT3 as assessed by Biacore SPR analysis and functional activity as assessed by DCTNFα production assay; however, substitution of the W101 residue resulted in a significant loss of binding as determined by Biacore SPR assay.

[0283] In summary, humanized 52B8 is an anti-ILT3 mAb (52B8 V H 1 M64V / V L 5 N34Q IgG4 S228P / κ), included in V L A frame replacement (M4L) in V H A framework substitution (A97G) in .

[0284] Example 4

[0285] The binding kinetics and affinity of anti-human ILT3 clones to recombinant proteins of human or macaque ILT3-His tags were measured by surface plasmon resonance using the Biacore T200 system (GE Healthcare, Piscataway, NJ). HBS-EP+ buffer (BR-1006-69) was used as running buffer. According to the manufacturer's instructions, anti-human Fc antibodies (Human Fc Capture Kit, BR100839, GE Healthcare) were fixed on Series SCM5 sensor chips (BR100530 or 29149603, GE Healthcare) by amine coupling chemistry In all four flow cells on. Flow cell 1 was used as a reference for background subtraction and was not used for capture. The anti-human ILT3 antibodies listed above (diluted to 1 μg / mL in HBS-EP+ buffer) were injected at 10 μL / mL for 10 seconds on the anti-human Fc capture surface in flow cells 2, 3 and 4, which resulted in antibody capture levels in the range of 60-70RU six points, ranging from 20nM to 0.31nM of a 2-fold dilution series of human or macaque ILT3-His protein and two zeros (HBS-EP+) at 50 μL / mL on the reference and capture antibody surface for 180 seconds of binding, followed by 600 seconds of dissociation. After each injection cycle, all four flow cells were regenerated using a 30-second second injection of 3M MgCl2 solution at a flow rate of 10 μL / min. In the Biacore T200 Evaluation Software (Version 2.0), the reference-deducted sensorgrams were 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).

[0286] Table 7 summarizes the binding kinetics and affinities of anti-human ILT3 antibodies to recombinant human or cynomolgus ILT3.

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294] Example 5

[0295] Chimeric anti-ILT3 52B8 mouse V that binds to human ILT3 H / Human IgG4(S228P): Mouse V L / Epitope mapping of human kappa ("c58B8"; mAb 73) by hydrogen-deuterium exchange (HDX) mass spectrometry

[0296] The contact area of ​​the antibody with the human ILT3 extracellular domain was determined by using hydrogen deuterium exchange mass spectrometry (HDX-MS) analysis. HDX-MS measures the incorporation of deuterium in the amide backbone of the protein, and changes in this incorporation are affected by the solvent exposure of hydrogen. A comparison of the deuterium exchange levels in samples of the antigen alone and the samples of the bound antibody was performed to determine the area on the ILT3 extracellular domain that can be contacted with the antibody. The human ILT3 extracellular domain with a C-terminal His tag (human ILT3-His) has the amino acid sequence shown in SEQ ID NO: 1.

[0297] The His-tagged human ILT3-His extracellular domain was pre-incubated with antibody c58B8 (mAb 73), a chimeric anti-ILT3 52B8 mouse V H M64V / Human IgG4(S228P): Mouse V L / human κ, which comprises a HC having an amino acid sequence of SEQ ID NO: 113 and a LC having an amino acid sequence shown in SEQ ID NO: 116, followed by incubation in deuterium buffer. Human ILT3-His and antibodies were buffer exchanged to PBS pH 7.4 using a 3k MWCO spin column. Human ILT3-His (80 pmol / μL) was mixed with an equal volume of antibody (40 pmol / μL), or as an unbound control, PBS pH 7.4. Before starting the labeling experiment, the samples of bound antibodies and unbound controls were incubated at room temperature for 1 hour.

[0298] In order to deuterium label the sample, 2 μL of sample was mixed with 25 μL of PBS in deuterium oxide, pH 7.6. The labeling time point was 30, 300, 3000, 6000 or 12000 seconds. After the set time, 25 μL of labeling mixture was added to 30 μL of cold quenching buffer (8M urea, 150mM 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 the pepsin / protease XIII column and the resulting peptide was loaded onto the capture column. After three minutes, the analytical gradient and mass spectrometer were started. By incubating 2 μL of human ILT3-His with 108 μL of deuterated denaturing buffer (4M urea, 150mMTCEP, in 99.5% of deuterium oxide), a fully deuterated sample was produced. The sample was incubated overnight at 37 ° C. Then 55 μL was injected directly into the column chamber and data was acquired.

[0299] LC-MS / MS data of unlabeled samples were collected and searched before deuterium labeling to verify successful digestion of proteins and generate peptide lists. Data were searched against databases using Proteome Discoverer 1.4 and the SEQUEST HT search algorithm (ThermoFisher Scientific). The protein database used was the human ILT3-His sequence linked to the yeast Saccharomyces cerevisiae database.

[0300] After labeling, a 55 μL sample aliquot was applied to a NovaBioAssays Pepsin / Protease XIII column, followed by chromatography on a Waters CSH C18 guard column and a Waters CSH C18 1x50mm 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: 8M urea, 150mM TCEP; labeling buffer: PBS, pH 7.6; blank buffer: PBS, pH 7.4. The mass spectrometer was a ThermoScientific Orbitrap-Elite. To measure the deuterium-labeled samples, the mass spectrometer was set to acquire a full scan MS data in the orbitrap at a resolving power 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 resolving power of 120,000, followed by ten data-dependent MS / MS spectra in the ion trap.

[0301] The LC system used was a Waters nano ACQUITY for analytical column gradients and a Waters 515 isocratic pump for sample digestion and loading. For digestion and loading of the samples, the buffer used was 2% acetonitrile and 0.1% trifluoroacetic acid at 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, followed by a 1.5 minute wash with 80% B and then a re-equilibration at 2% B for 3 minutes. The column was then washed by cycling the gradient between 2% and 80% B three times, 1 minute per step, followed by a final equilibration at 2% B for 5 minutes. The trapping 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.

[0302] Deuterium labeled sample processing was done with a Leaptec H / DX 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 compartments were set to 1.5°C. The immobilized pepsin column (Pepsin / Protease XIII column NBA2014002, 2.1x30mm, NovaBioAssay) was kept outside the column compartment at room temperature.

[0303] Deuterium-labeled differential thermogram of the antibody-bound human ILT3-His amino acid residues is shown in Figure 3A HDX mass spectrometry showed that the antibodies disclosed herein and other antibody families that cross-compete with the antibodies bind to an epitope that comprises or consists of at least one amino acid in 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 the three-dimensional surface structure model of the human ILT3 extracellular domain are shown, showing protected amino acid residues. These protected amino acid residues contain split or discontinuous epitopes that span the boundary between the D1 and D2 domains of the extracellular domain. Figure 3CIt is a ribbon diagram showing the position of the epitope on the extracellular domain of human ILT3. The black residues are protected from being labeled by antibodies. The white residues are not shown in the changes in labeling, and the dark gray residues are not obtained from it. The deuterium labeling difference of each residue is averaged and mapped to the crystal structure of ILT3 (Cheng et al., "Crystal structure of leukocyte Ig-like receptor LILRB4 (ILT3 / LIR-5 / CD85k): amyeloid inhibitory receptor involved in immune tolerance." J Biol Chem 286: 18013-25 (2011)).

[0304] Similar HDX mapping experiments were performed using antibodies ZM4.1, DX439, DX446, and 9B11. The ZM4.1 antibody 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 U.S. Patent 7,777,008. Of 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, classification studies 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 heat maps of the binding of antibodies ZM4.1, DX439, DX446, and 9B11 to human ILT3.

[0305] Example 6

[0306] Chimeric anti-ILT3 52B8 mouse V H / Human IGg4(S228P): Mouse V L Pharmacokinetics of human κ ("c58B8"; mAb 73) in NSG mice

[0307] Evaluation of chimeric anti-ILT3 52B8 mouse V in the Panc08.13 human-NSG mouse model and the SK-MEL-5 human CD34+-NSG mouse model H / Human IgG4(S228P):Mouse V L / Pharmacokinetics of human κ (c85B8; mAb 73).

[0308] SK-MEL-5 is a human melanoma-derived cell line that can grow as subcutaneous tumors. Panc 08.13 is a human pancreatic cancer-derived tumor cell line. The Panc 08.13 human-NSG model has been shown to be sensitive to pembrolizumab and ipilimumab treatment. The SK-MEL-5 model has a robust and diverse myeloid infiltration in the tumor compared to the Panc 08.13 model. Both models show increased ILT3 expression on human CD14+ myeloid cells in the tumor and spleen.

[0309] 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 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 plates. The assay was qualified, with an LLOQ of 40 ng / mL and an MRD of 100 for the assay.

[0310] In the Panc08.13 hu-NSG mouse model, 20 mg / kg of the antibody was administered IP 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 the 5th and 6th days after the fourth dose. In the SK-MEL-5 huCD34+-NSG mouse model, the antibody was administered by IP at 2 and 20 mg / kg per week. Blood samples were collected before the third dose (Ctrough) and 24h (Cmax) after the third dose. Terminal blood samples were also collected on the 3rd and 7th days after the third dose. Free (unbound) antibody concentrations were determined by antigen capture assay.

[0311] Pharmacokinetic parameters were generated from historical IgG4 antibody data (humanized IgG4 antibody 1, 3, 10, 30 mg / kg administered as iv bolus in C57BL / 6J mice) and Phoenix NLME.PK curves for the studied doses of antibody were simulated based on the generated pharmacokinetic parameters.

[0312] PK analysis of historical IgG4 antibody data showed a linear relationship between AUC and the dose studied (see Figure 4). The PK profiles of c52B8 at the doses studied were simulated based on historical IgG4 antibody data under assumptions including linear PK of c52B8 across the different tested doses, no PK differences between different mouse strains, rapid absorption of the antibody after IP administration, and 100% bioavailability. The results showed that the simulated curves at 20 mg / kg followed the observed c52B8 concentrations in both the Panc08.13 human-NSG model and the SK-MEL-5 huCD34+-NSG model.

[0313] Example 7

[0314] Anti-ILT3 monoclonal antibodies activate dendritic cells and reduce the suppressive capacity of myeloid-derived suppressor cells (MDSCs)

[0315] Freeze, thaw human PBMC separated from fresh leukocytes, and purify CD14+ monocytes by negative selection. Purified cells were cultured for 5 days with GM-CSF (1000U / mL) and IL4 (1000U / mL). These immature DCs were then further cultured for 42 hours in the presence of IL-10 (50ng / mL) and LPS (1ug / mL) with or without anti-ILT3 antibodies. TNFα was measured in the culture supernatant.

[0316] Titration experiments showed that c52B8, when added during the polarization step, caused a dose-dependent increase in TNFα secretion in the culture medium, whereas control IgG4 did not (the control was a variant of a commercially available antibody against RSV, trade name Synagis) ( Figure 5A The antibody concentration required to produce a half-maximal increase in TNFα levels (EC50) was approximately 1.9 ng / mL. H and V L This is no different for chimeric variants with Fc fusions of human IgG1 frameworks (mAb 78) or human IgG1 frameworks (mAb 76) with N297A mutations. These data suggest that Fc receptor binding does not play any role in functional activity in this assay. Independence relative to Fc receptor binding controls the possibility that the activation mechanism in this assay is DCs that become activated by recognizing other DCs in cultures decorated with antibodies, which would be a mechanism unrelated to ILT3.

[0317] Figure 5B and 5C Shown in two donors in c52B8 (mAb 73) and humanized anti-ILT3 mAb 52B8 V H 1M64 V / V LThere were no significant differences in functional activity between 5 N34Q IgG4 S228P / κ (mAb 46). As shown, when antibody c52B8 was added during polarization of DCs, but not during T cell priming, DCs were better able to activate T cells to proliferate, similar to IL10-intolerant DCs. 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 chimera 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 expected if humanized mAb 46 were used.

[0318] Example 8

[0319] Anti-ILT3 antibodies reduce the suppressive capacity of myeloid-derived suppressor cells (MDSCs)

[0320] Without being ascribed to any particular theory or hypothesis, we hypothesize that productive T cell responses to tumors can, in certain cases, be limited by the presence of immature and suppressive myeloid cells. These cells express ILT3, which we hypothesize to act in an inhibitory manner to maintain an immature state characterized by low HLA-DR expression, IL-10 production, and effective inhibition of T cell activation and proliferation. The establishment of a model based on in vitro co-culture of human PBMCs with SKMEL5 tumor cells, followed by purification of MDSCs and testing their ability to inhibit the proliferation of autologous CD8+ T cells, enabled the exploration of this aspect of ILT3 biology. This example demonstrates that c52B8 and humanized 52B8 (mAb 46) are able to impair the acquisition (or maintenance) of a T cell suppressive phenotype.

[0321] To generate MDSC, healthy human PBMCs were cultured with SKMEL5 cells and 20ng / mL of GM-CSF for 7 days. CD33+ cells were collected by magnetic bead selection based on positive antibodies, and then co-cultured with purified autologous CD8+T cells at the indicated ratio for 3 days in the presence of polyclonal stimulants. In both co-culture and T cell inhibition steps, the culture contained c52B8 (mAb 73), humanized 52B8 (mAb 46), or isotype control antibodies (1 μg / mL). T cell inhibition assays were performed with a ratio of T cells to MDSC of 4:1, and the amount of interferon gamma (INFγ) produced was measured.

[0322] Fig. 6A and Figure 6BThe activity of humanized 52B8 and c52B8 in the MDSC model at a ratio of T cells to MDSCs is exemplified, where the effect of these antibodies was most pronounced, indicating that these antibodies reduced the suppressive capacity of MDSCs in a comparable manner. These data further indicate that the data generated with c52B8 are representative of what would be found using humanized mAb 46.

[0323] Example 9

[0324] Anti-ILT3 antibody cC52B8 inhibits SK-MEL-5 tumor growth in SK-MEL-5hu-NSG mice bearing SK-MEL-5 subcutaneous tumors

[0325] Weekly systemic administration of c52B8 to mice bearing established subcutaneous tumors provided inhibition of tumor growth ( Figure 7 ). Animals were randomly assigned to treatment groups based on tumor volume on day 21 after implantation and were administered 20 mg / kg c52B8 (mAb 73) or isotype control subcutaneously once a week starting on day 21. The data shown in the left figure are mean and std. error (nine animals per group). The right figure shows individual animal tumor growth curves. Body weight decreased to a similar extent in both the control and 52B8 groups. This study represents three independent studies.

[0326] The extent of inhibition of tumor growth was consistent and similar across three independent studies and closely resembled the effect of anti-ILT4. Other mechanisms tested to date (e.g., anti-PD-1, anti-ILT4, anti-CD27, anti-GITR) have not provided regression, leading us to speculate that tumor stasis may represent the basis of this model. This is clearly different from the mouse syngeneic models commonly used for preclinical efficacy assays.

[0327] Example 10

[0328] Immune activation in SK-MEL-5hu-NSG after c52B8 treatment

[0329] To understand the immune mechanisms mediating tumor efficacy, tumor infiltrating immune cells were analyzed and sHLA-G levels were measured in the blood. Mice were treated with c52B8 (2 and 20 mg / kg ip QW). Based on the C detected in the mini-PK and simulations using historical studies max and C troughThe antibody dose was selected based on the level of sHLA-G. 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. Thirty percent tumor growth inhibition was observed in mice treated with 20mpk 52B8. However, due to the large variability associated with the humanized tumor model, no statistically significant differences were detected. The moderate tumor efficacy of 52B8 was associated with a moderate reduction in tumor CD4+CD127-CD25+ T suppressor cells (21% vs. 14%) and blood sHLA-G levels, as well as an increase in the activation of T cells in the tumor (CD69 intensity, 14 vs. 23). As shown in Figure 8, no cytokine changes were detected for c52B8 treatment.

[0330] Embodiment 11

[0331] Effect of the anti-ILT3 antibody c52B8 combined with pembrolizumab in the Panc08.13 hu-NSG model: tumor efficacy and immune activation

[0332] The 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 shown in Figure 2. Fig. 9A , Fig. 9B , Figure 9C and Fig.9D shown.

[0333] Example 12

[0334] Effect of the combination of anti-ILT3 antibody 52B8 and pembrolizumab in MDSC / T cell suppression assay

[0335] In the MDSC / T cell inhibition assay, humanized anti-ILT3 antibody 52B8 (mAb 46) with and without pembrolizumab achieved increased T cell activity. When mAb 46 was used in combination with pembrolizumab, the effect was additive.

[0336] In order to generate MDSC, healthy human PBMC from a specific donor was cultured with SKMEL5 cells and 20ng / mL of GM-CSF for seven days. Cultures were treated with 52B8 (1 μg / mL) or isotype control antibodies (1 μg / mL). CD33+ cells were collected with anti-CD33 magnetic microbeads and LS column separation (Miltenyi Biotec, Germany), and then co-cultured with purified autologous CD8+ T cells at a specified ratio for 3 days in the presence of polyclonal stimulants. Autologous CD8+ T cells were separated from healthy human PBMC using magnetic beads based on negative antibodies (Stem Cell Technologies, Canada), and then co-cultured with CD33+ myeloid cells in 96-well plates at a ratio of 8:1 (T cells: MDSC) for 2 days. In both co-culture and T cell inhibition steps, cultures included humanized 52B8 (mAb 46) or isotype control antibodies (IgG4) (1 μg / mL) alone or in combination with pembrolizumab (2 μg / mL). The total antibody concentration in each treatment was adjusted to 3ug / mL with an isotype control antibody. T cell proliferation was induced by polyclonal stimulatory anti-CD3 / CD28 beads and IL2. IFNγ levels were determined in culture supernatants using MSD ELISA (Mesoscale Discovery, MD). T cell suppression assays were performed using a 4:1 or 8:1 T cell to MDSC ratio and the amount of interferon gamma (INFγ) produced was measured. The results are shown in Figure 10-14 middle.

[0337] Fig.10 It was shown that in an MDSC / T cell suppression 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 suppressive capacity of MDSCs to an extent comparable to that of the chimeric anti-ILT3 antibody c52B8 (mAb 73; lot 26AVY).

[0338] Figure 11-14 Shown are MDSC / T cell suppression assays using (a) MDSCs obtained from PBMCs from human donor D001003835 at a T cell to MDSC ratio of 4:1 or 8:1 ( Fig.11 ); (b) using MDSCs obtained from PBMCs from human donor D001003180 at a ratio of 4:1 or 8:1 MDSC to T cells ( FIG. 12 ); (c) using MDSCs obtained from PBMCs from human donor D001003507 at a ratio of 4:1 or 8:1 T cells to MDSCs ( Fig.13); and using MDSCs obtained from PBMCs from human donors at a T cell to MDSC ratio of 8:1 ( Fig.14 ), humanized anti-ILT3 antibody 52B8 (mAb 46) combined with pembrolizumab to reduce MDSC inhibition of T cell activation at a higher level than each alone. The results are summarized in Tables 8 and 9. Figure 10-13 As shown in Tables 8 and 9, the combination of anti-ILT3 antibody 52B8 with pembrolizumab produced an additive effect that increased the activation of T cells relative to that achievable with either pembrolizumab or 52B8 alone. As shown, the increase in IFNγ for the combination ranged from 41% to 74% relative to the other treatments. These results suggest that the combination of pembrolizumab with 52B8 does not result in an excessive or uncontrolled increase in T cell activation.

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[0342] Example 13

[0343] Effect of the anti-ILT3 antibody 52B8 combined with pembrolizumab in mixed lymphocyte reactions of polarized IL-10 DCs and allogeneic CD8+ T cells

[0344] In this example, mixed lymphocyte reactions of IL-10 polarized human monocyte-derived dendritic cells and allogeneic CD8+ T cells were cultured for 4 days, followed by measurement of interferon gamma (IFNγ) in the culture supernatant as a readout of T cell activation. In this experiment, the activity of pembrolizumab, 52B8, or a combination of both was compared to an isotype control antibody (IgG4 in both cases) in nine allogeneic donor pairs.

[0345] 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 from three donors were isolated and mixed leukocyte reactions (MLRs) (30k DCs vs 150k CD8+ T cells) were established in a 96-well format from these three donors at a DC:T cell ratio of 1:5, with and without IgG4 (lot 92ASJ); cells were treated with and without 2 μg / mL of pembrolizumab (lot 42ASN). IgG4 or 52B8 were also added back to the MLR at 1 μg / mL. Ending with 9 MLR pairs of IL10 DC:CD8+ T cells:

[0346] DC129 vs T30, T3788 and T3259

[0347] DC226 vs T30, T3788 and T3259

[0348] DC196 vs T30, T3788 and T3259

[0349] IFNγ supernatants were collected on day 4 and quantified using Meso Scale Discovery (MSD). Additional supernatant fractions were collected on day 5, and cells were collected and stained for PD1 and PDL1 expression. Dendritic cell staining was performed on day 7 of differentiation (just before MLR setup). T cell staining of CD8+ T cells on day 5 was performed for MLR assays.

[0350] Fig.15 Results are shown for all donor pairs merged into one graph (each label is a donor pair). As shown, combining 52B8 with pembrolizumab achieved reversal of T cell tolerance, resulting in a statistically significant increase in activation of CD8+ T cells.

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[0374] Although the present invention is described herein with reference to the embodiments shown, it should be understood that the present invention is not limited thereto. Those with ordinary skills in the art and who can obtain the teachings herein will recognize other modifications and embodiments within the scope thereof. Therefore, the present invention is limited only by the claims appended hereto.

Claims

1. An antibody that binds to human immunoglobulin-like transcript 3 (ILT3), comprising: (a) a heavy chain (HC) complementarity determining region (HC-CDR1) with an amino acid sequence as set forth in SEQ ID NO: 17; an HC-CDR2 with an amino acid sequence as set forth in SEQ ID NO: 19, 20 or 21; and an HC-CDR3 with an amino acid sequence as set forth in SEQ ID NO: 23; and (b) a light chain (LC) complementarity determining region (LC-CDR1) having an amino acid sequence as shown in SEQ ID NO:34, 35, 36, 37, 38, 39, 40, 41 or 42; a LC-CDR2 having an amino acid sequence as shown in SEQ ID NO:43; and a LC-CDR3 having an amino acid sequence as shown in SEQ ID NO:

44.

2. The antibody according to claim 1, wherein (a) the amino acid sequence of the HC-CDR1 is shown in SEQ ID NO: 17; the amino acid sequence of the HC-CDR2 is shown in SEQ ID NO: 20; and the amino acid sequence of the HC-CDR3 is shown in SEQ ID NO: 23; and (b) the amino acid sequence of the LC-CDR1 is shown in SEQ ID NO:41; the amino acid sequence of the LC-CDR2 is shown in SEQ ID NO:43; and the amino acid sequence of the LC-CDR3 is shown in SEQ ID NO:

44.

3. The antibody according to claim 1 or 2, wherein the V H Contains selected from human V H 1. V H 2. V H 3. V H 4. V H 5. and V H 6, and the framework of 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 Contains 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. V λ 10, 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.

4. The antibody of claim 1 or 2, wherein the antibody comprises a 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 a combination thereof compared to the amino acid sequence of a native IgG1, IgG2, IgG3 or IgG4 isotype constant domain.

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

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

7. The antibody according to claim 3, wherein the antibody comprises V and V having the amino acid sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively. H and V L .

8. The antibody according to claim 6, wherein the antibody comprises a V having an amino acid sequence as shown in SEQ ID NO:

118. H and V having the amino acid sequence shown in SEQ ID NO: 140 L .

9. The antibody according to claim 6, wherein the antibody comprises a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO: 143 and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO: 165, and variants thereof wherein the HC lacks a C-terminal lysine residue or a C-terminal glycine-lysine.

10. A composition comprising: The antibody according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

11. A nucleic acid molecule encoding the anti-ILT3 antibody according to any one of claims 1 to 9. A vector comprising the nucleic acid molecule according to claim 11 . A host cell comprising the vector according to claim 12 .

14. A method for producing an antibody according to any one of claims 1 to 9, comprising: (a) providing a host cell comprising the host cell of claim 13; (b) culturing the host cell in a culture medium under conditions capable of expressing the antibody encoded by the vector; and, (c) obtaining the antibody from the culture medium to provide the antibody.

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