Humanized antibodies against Nectin-2 and drug conjugates thereof

Abstract

The present invention provides humanized monoclonal antibodies that recognize human Nectin-2 with high affinity and specificity and inhibit its binding to CD112R. These antibodies carry cytotoxic payload and do not interact with Fc gamma receptors. The present invention further provides pharmaceutical compositions comprising the antibodies and methods for their use in cancer immunotherapy.

Claims

1. A conjugate, comprising: (a) an antibody that binds Nectin-2 or an antigen-binding fragment thereof that comprises: A) a heavy chain variable region (VH) comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 11 and (i) a complementarity determining region (CDR) 1 having an amino acid sequence according to SEQ ID NO: 1; (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 2; and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 3; and B) a light chain variable region (VL) comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 12 and (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 4; (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 5; and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 6; and (b) a topoisomerase I inhibitor conjugated to the Nectin-2 antibody or antigen-binding fragment thereof using a linker.

2. The conjugate of claim 1, wherein the CDR2 of the VH has an amino acid sequence according to SEQ ID NO: 41.

3. The conjugate of claim 1, wherein the CDR1 of the VL has an amino acid sequence according to SEQ ID NO: 43.

4. The conjugate of claim 1, wherein the CDR2 of the VH has an amino acid sequence according to SEQ ID NO: 41, and the CDR1 of the VL has an amino acid sequence according to SEQ ID NO: 43.

5. A pharmaceutical composition comprising the conjugate of claim 1, and a pharmaceutically acceptable excipient, carrier, or diluent.

6. The conjugate of claim 1, wherein the topoisomerase I inhibitor is DXd.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A-1C depict the correlation of Nectin-2 mRNA levels (high or low as indicated) with survival probability of Low-grade glioma (FIG. 1A), Kidney Renal Clear Cell Carcinoma (FIG. 1B) and lung adenocarcinoma (FIG. 1C) patients. Data sets were obtained from the TCGA site and analyzed using oncolnc.org site (https://doi.org/10.7717/peerj-cs.67). N depicts number of patients included at the analysis.

(2) FIG. 2 is a schematic illustration of receptors expressed on immune cells and their respective affinities to Nectin-2 (CD112), which is expressed by tumors or on antigen presenting cells (APCs). TIGIT is a co-inhibitory receptor on many immune cells (e.g., T and NK cells); DNAM-1 (also termed CD226) is an activating receptor on many immune cells (e.g., T cells), and CD112R (also termed PVRIG) and TIGIT are co-inhibitory receptors on lymphoid immune cells (e.g., T and NK cells); Nectin-2 acts as an inhibitory ligand for immune cells, mainly via its high-affinity binding to CD112R (rectangle).

(3) FIG. 3 is a graph showing percent of tumors positive for Nectin-2 expression. Data obtained from proteinatlas.com using the HPA012759 mAb (anti Nectin-2; Sigma-Aldrich). In 17/20 indications moderate-high membranous expression of Nectin-2 is seen.

(4) FIGS. 4A-4C depict improved on-cell binding of the humanized variants of the parental (chimeric) anti-Nectin2 mAb. Nectin-2 expressing cells were analyzed by FACS analysis for binding of serially diluted mAb humanized variants. EC-50 values were calculated for each variant and are reported relative to the EC-50 value of the chimeric Ab (HOKO), which was set as 1. FIG. 4A-fold change EC-50 binding to 293T cells expressing human Nectin-2. FIG. 4B-fold change EC-50 binding to Vero cells derived from African green monkey (AFG, Chlorocebus) and naturally expressing Nectin-2 (XP_007995342.1). FIG. 4C-results of CD112R blocking by the different humanized mAb variants, as assessed by FACS analysis of CD112R-Fc binding to CHO(K)1 cells overexpressing human Nectin-2, in presence of the anti-Nectin-2 humanized clones in concentrations from 66-0.81 nM. Variants maintaining >70% blocking capacity at the lowest dose were considered superior.

(5) FIGS. 5A-5C depict the killing effect of humanized anti-Nectin-2 ADC variants. ADC was based on Saporin (ZAP) and the A549 target cells (lung adenocarcinoma) were confirmed to express Nectin-2. FIG. 5A shows the superior killing effect of the lead clone H3K3, compared to other humanized variants tested at two concentrations. FIG. 5B depicts comparable levels of ADC activity between the humanized H3K3 and H4K2 variants. FIG. 5C-comparable levels of ADC activity between the chimeric mAb (HOKO), and the humanized H3K3 variant at three concentrations. H3K3 was also tested when grafted on IgG2-P238H, which has an FcgR-null Fc, and which had no effect on the potency of this variant. All killing results are significant (p<0.001, two-way t-test), unless indicated by NS (not significant).

(6) FIGS. 6A-6C demonstrate that humanized H3K3-FcgR.sup.null anti-Nectin-2 ADCs, conjugated to various toxins, lead to robust killing of various solid tumor cell lines. The Figures show significant killing activity of the H3K3-FcgR.sup.null ADCs, at different ADC concentrations and across different cancer targets after 72 hours of incubation. FIG. 6A depicts killing of RKO cells (colorectal adenocarcinoma). FIG. 6B depicts killing of MDA-MB-231 cells (triple negative breast cancer). FIG. 6C depicts killing of A549 cells (lung adenocarcinoma). Killing of target cells was significant (p<0.001), unless indicated by NS (not significant). While all ADCs had a toxic effect, its magnitude varied pending the conjugated toxin and nature of target cells. Still, the ADCs containing either MMAE or MMAF were superior to all others across all target cells.

(7) FIG. 7 demonstrates that humanized H3K3-FcgR.sup.null anti-Nectin-2 ADCs are not internalized via interaction with the high affinity FcgR-CD64. CHO-K1 cells or CHO-K1 cells overexpressing human CD64 (hCD64), but not Nectin-2, were incubated with two versions of H3K3-FcgR.sup.null ADCs (conjugated to either MMAE or DM4), or with control irrelevant ADCs that have either hlgG4 (S228P) or hlgG1 Fc (both conjugated to DM4), at 12 ug/ml for 72 hours. Specific increased killing was seen for the CD64 positive cells when either hlgG4 or hlgG1 was used, but not when the FogRnull Fc H3K3 ADC variants were used. Note that background, non-specific killing was induced by DM4, regardless of either Nectin-2 or CD64 expression by the target cells.

(8) FIG. 8 demonstrates that humanized H3K3-FcgR.sup.null anti-Nectin-2 ADCs lead to robust killing of a hematological cell line. H3K3-FcgR.sup.null Abs, with the indicated linker-payload combinations, were incubated at concentration from 9-1 mg/ml, for 72 hours with HL-60 cells (an AML model). All linker-payload combinations, except DM1, resulted in >90% killing at the high dose and significant killing at all doses tested (p<0.001).

(9) FIGS. 9A-9B depict in-vivo efficacy of the humanized H3K3-FcgR.sup.null anti-Nectin-2 ADCs against the hematological tumor cells line, HL-60 implanted s.c. to Nude female mice. FIG. 9A compares the effect of treatment with different combinations of linker payloads (LP). Two of the LP combinations had a significant effect: DM4 resulted in significant tumor growth inhibition (TGI, p<0.05), while complete tumor regression was seen for MMAE. The effect of the MMAE-based ADC is shown in FIG. 9B without including the other treatments.

(10) FIG. 10 depicts in-vivo efficacy of the humanized H3K3-FcgR.sup.null anti-Nectin-2 ADC (1107-MC-VC-MMAE) against the solid (colorectal adenocarcinoma) tumor cell line, RKO. An empty vehicle was used as control.

(11) FIG. 11 depicts in-vitro killing of MDA-MB-231 (triple negative breast cancer or TNBC) cells by the humanized H3K3-FcgR.sup.null anti-Nectin-2 mAb (NTX1107) linked to Dxd, compared to Trodelvy (Sacituzumab govitecan), an approved ADC drug for TNBC. Both ADCs use the same class of cytotoxic payload (i.e., topoisomerase I (TOP1) inhibitors). MDA-MB-231 cells were incubated in presence of the indicated concentration of these ADCs. Killing was evaluated after 120 hours. Robust killing of target cells was induced by both ADCs with significant superiority of Trodelvy at both concentrations (*** p<0.0005).

(12) FIG. 12 depicts in-vivo efficacy of humanized H3K3-FcgR.sup.null anti-Nectin-2 ADC (NTX1107), compared to Trodelvy, against MDA-MB-231 s.c. tumor model in Nude female mice. NTX1107-Dxd was able to significantly inhibit tumor growth, resulting in tumor stasis, while Trodelvy had no effect on tumor growth at the same treatment regimen.

(13) FIG. 13 compares the effect of in-vivo treatment with H3K3-FcgR.sup.null-Dxd (NTX1107-Dxd) to that of H3K3-FcgR.sup.null MMAE (NTX1107-MMAE) against MDA-MB-231 s.c. tumor model in Nude female mice. Complete tumor regression was seen for H NTX1107-MMAE with 7/7 mice having no measurable tumors at the end of the study. Tumor regression was seen for NTX1107-Dxd with 4/7 mice having no measurable tumors at the end of the study.

DETAILED DESCRIPTION OF THE INVENTION

(14) The present invention provides humanized monoclonal antibodies that recognize Nectin-2. Advantageously, the antibodies of the invention are almost fully humanized, thus avoiding the risk of adverse immune response towards the antibodies and are therefore likely to be safe for use in humans. The antibodies of the invention are characterized by having unique CDR sequences and novel humanized framework sequences and design.

(15) The present invention further provides in some embodiments antibody-drug conjugates, or ADCs, comprising the humanized antibodies described herein, which are useful in treating cancer.

(16) In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the embodiments provided may be practiced without these details. Unless the context requires otherwise, throughout the specification and claims which follow, the word comprise and variations thereof, such as, comprises and comprising are to be construed in an open, inclusive sense, that is, as including, but not limited to. As used in this specification and the appended claims, the singular forms a, an, and the include plural referents unless the content clearly dictates otherwise. It should also be noted that the term or is generally employed in its sense including and/or unless the content clearly dictates otherwise. Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

(17) As used herein the term about refers to an amount that is near the stated amount by 10% or less.

(18) The term Nectin-2 or Nectin Cell Adhesion Molecule 2, as used herein refers to a human plasma membrane glycoprotein, also known as CD112, and PVRL2. The Nectin-2 protein is a single-pass type I membrane glycoprotein with two Ig-like C2-type domains and an Ig-like V-type domain. This protein is one of the plasma membrane components of adherent junctions. It also serves as an entry for certain mutant strains of herpes simplex virus and pseudorabies virus, and it is involved in cell to cell spreading of these viruses. An exemplary Nectin-2 according to the invention is set forth in SwissPort, UniPort and GenBank symbols or accession numbers: Gene ID: 5819, Q92692, 168093, NP_001036189.1, NP_002847.1, and #Q92692.

(19) According to an aspect, the present invention provides a humanized antibody that specifically bind Nectin-2, or a fragment thereof comprising at least the antigen binding site, wherein the antibody or a fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; and wherein the light chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.

(20) According to another aspect, the present invention provides a humanized antibody that specifically binds Nectin-2, or a fragment thereof comprising at least the antigen binding site, wherein the antibody, or a fragment thereof, comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: (i) a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 1-3; and (ii) a set of four heavy chain framework sequences, wherein: FR-H1 (frame work heavy chain No. 1) is selected from the group consisting of SEQ ID NOs: 21, 25, and 27; FR-H2 is SEQ ID NO: 22; FR-H3 is selected from the group consisting of SEQ ID NOs: 23, 26, 28, and 29; FR-H4 is SEQ ID NO: 24; and the light chain variable region comprising: (i) a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 4-6; and (ii) a set of four light chain framework sequences, wherein FR-L1 (frame work light chain No. 1) is selected from the group consisting of SEQ ID NOs: 30, 34, 37, and 39; FR-L2 is selected from the group consisting of SEQ ID NOs: 31 and 35; FR-L3 is selected from the group consisting of SEQ ID NOs: 32, 36, 38, and 40; and FR-L4 is SEQ ID NO: 33.

(21) The frameworks are the non-CDR sequences of the chain variable regions. FR-H1 is the sequence before CDR1 in the heavy variable chain, FR-H2 is the sequence between CDR1 and CDR2, FR-H3 is the sequence between CDR2 and CDR3, and FR-H4 is the sequence after CDR3. FR-L1 is the sequence before CDR1 in the light variable chain, FR-L2 is the sequence between CDR1 and CDR2, FL-H3 is the sequence between CDR2 and CDR3, and FR-L4 is the sequence after CDR3.

(22) According to an additional aspect, the present invention provides a humanized antibody or antigen binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: i. a set of three CDR sequences, the CDRs comprising the sequences SYW, SEQ ID NO: 8, and SEQ ID NO: 3; and ii. a set of four heavy chain framework sequences, wherein: FR-H1 is selected from the group consisting of SEQ ID NOs: 21, 25, and 27; FR-H2 is SEQ ID NO: 22; FR-H3 is selected from the group consisting of SEQ ID NOs: 23, 26, 28, and 29; FR-H4 is SEQ ID NO: 24; and the light chain variable region comprising: i. a set of three CDR sequences, the CDRs comprising the sequences set forth in SEQ ID NO: 10, SAS, and SEQ ID NO: 6; and ii. a set of four light chain framework sequences, wherein: FR-L1 is selected from the group consisting of SEQ ID NOs: 30, 34, 37, and 39; FR-L2 is selected from the group consisting of SEQ ID NOs: 31 and 35; FR-L3 is selected from the group consisting of SEQ ID NOs: 32, 36, 38, and 40; and FR-L4 is SEQ ID NO: 33.

(23) According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain variable region comprising a set of three CDR sequences, the CDRs comprising the sequences SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9; and a light chain variable region comprising a set of three CDR sequences, the CDRs comprising the sequences SEQ ID NO: 10, SAS, and SEQ ID NO: 6.

(24) Among the provided antibodies are monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies and polyreactive antibodies), and antibody fragments. The antibodies include antibody-conjugates and molecules comprising the antibodies, such as chimeric molecules. Thus, an antibody includes, but is not limited to, full-length, as well as fragments and portion thereof retaining the binding specificities thereof, such as any specific binding portion thereof including those having any number of, immunoglobulin classes and/or isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE and IgM); and biologically relevant (antigen-binding) fragments or specific binding portions thereof, including but not limited to Fab, F(ab) 2, Fv, and scFv (single chain or related entity). A monoclonal antibody is generally one within a composition of substantially homogeneous antibodies; thus, any individual antibodies comprised within the monoclonal antibody composition are identical except for possible naturally occurring mutations that may be present in minor amounts. A polyclonal antibody is a preparation that includes different antibodies of varying sequences that generally are directed against two or more different determinants (epitopes). The monoclonal antibody can comprise a human IgG1 constant region. The monoclonal antibody can comprise a human IgG4 constant region.

(25) The term antibody herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen binding (Fab) fragments, F(ab) 2 fragments, Fab fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (sFv or scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term antibody should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD. The antibody can comprise a human IgG1 constant region. The antibody can comprise a human IgG4 constant region.

(26) CDR identification or determination from a given heavy or light chain variable sequence, is typically made using one of few methods known in the art. For example, such determination is made according to the Kabat (Wu T. T and Kabat E. A., J Exp Med, 1970; 132:211-50) and IMGT (Lefranc M-P, et al., Dev Comp Immunol, 2003, 27:55-77).

(27) When the term CDR having a sequence, or a similar term is used, it includes options wherein the CDR comprises the specified sequences and also options wherein the CDR consists of the specified sequence.

(28) The antigen specificity of an antibody is based on the hyper variable region (HVR), namely the unique CDR sequences of both light and heavy chains that together form the antigen-binding site.

(29) Among the provided antibodies are antibody fragments. An antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab, Fab-SH, F(ab) 2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv or sFv); and multispecific antibodies formed from antibody fragments. In particular embodiments, the antibodies are single-chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFvs.

(30) A humanized antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all framework region (FR) amino acid residues are derived from human FRs. A humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody. A humanized form of a non-human antibody refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. According to some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

(31) The amino acid residues in the Fc domain can be substituted to be null, meaning the Fc domain does not bind Fc receptors or can bind with such low affinity and/or avidity as to not cause any Fc receptor signaling as a result of binding. The Fc domain can be null for binding to Fc receptors. Some example Fc receptors for which the Fc domain can be null for binding can be, but not limited to, FcRI (CD64), FcRIIA (CD32a), FcRIIB (CD32b), FcRIIIA (CD16a), FcRIIIA (CD16a) F158 variant, FcRIIIA (CD16a) V158 variant, or FcRIIIB (CD16b). The Fc domain may have one or more, two or more, three or more, or four or more amino acid substitutions that decrease binding of the Fc domain to an Fc receptor.

(32) According to some embodiments, the humanized antibody has a mutated Fc domain that prevents FcR-mediated internalization.

(33) According to some embodiments, the humanized antibody comprises a Fc null domain. According to certain embodiments, the Fc domain is null for binding to a Fc receptors.

(34) As used herein, an Fc null refers to a domain that exhibits weak to no binding to one or more of the Fc receptors.

(35) Antibody-Drug Conjugates

(36) According to an aspect, the present invention provides a conjugate comprising the humanized antibody as described herein fused to a toxin.

(37) According to some embodiments, the conjugate comprises an antibody or fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; and wherein the light chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.

(38) According to some embodiments, the toxin is selected from the group consisting of microtubule inhibitor, DNA synthesis inhibitor, topoisomerase inhibitor, and RNA polymerase inhibitor. Each possibility represents a separate embodiment of the invention.

(39) According to certain embodiments, the toxin is a microtubule-destroying drug. According to certain exemplary embodiments, the toxin is auristatin or a derivative thereof. According to certain embodiments, the auristatin derivative is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

(40) According to some embodiments, the toxin is saponin.

(41) According to some embodiments, the toxin is a maytansine derivative. According to certain embodiments, the maytansine derivative is DM4 or DM1.

(42) According to some embodiments, the toxin is quinoline alkaloid. According to certain embodiments, the quinoline alkaloid is SN-38.

(43) According to additional embodiments, the toxin is selected from the group consisting of MMAE, MMAF, Saporin, DM4, DM1, SN-38, Calicheamicin, DXd, PBD, Duocarmycin, Sandramycin, alpha-Amanitin, Chaetocin, Daunorubicin, 17-AAG, Agrochelin A, Doxorubicin, Methotrexate, Colchicine, Cordycepin, Hygrolidin, Herboxidiene, Ferulenol, Curvulin, Englerin A, Taltobulin, Triptolide, Cryptophycin, and Nemorubicin. Each possibility represents a separate embodiment of the invention.

(44) According to some embodiments, the toxin is a DNA topoisomerase I (TOP1) inhibitor. According to certain embodiments, the DNA topoisomerase I (TOP1) inhibitor is DXd.

(45) The toxins names are used herein as known in the art. Non limiting examples of suitable payloads include: DM1a N.sup.2-deacetyl-N.sup.2-(3-mercapto-1-oxopropyl)-maytansinea tubulin inhibitor. DM4a N.sup.2-deacetyl-N.sup.2-(4-mercapto-4-methyl-1-oxopentyl)-Ravtansinea tubulin inhibitor. SN-38a potent DNA topoisomerase I inhibitor, a member of the class of pyranoindolizinoquinolines that is (4S)-pyrano[3,4: 6,7]indolizino[1,2-b]quinoline-3,14-dione bearing two additional ethyl substituents at positions 4 and 11 as well as two additional hydroxy substituents at positions 4 and 9. DXdan exatecan derivative, a potent DNA topoisomerase I inhibitor (For example Cat. No.: HY-13631D of MCE). MMAFa monomethyl auristatin Fa tubulin inhibitor, having the formula C.sub.39H.sub.65N.sub.5O.sub.8. MMEAa monomethyl auristatin Ea tubulin inhibitor, having the formula C.sub.39H.sub.67N.sub.5O.sub.7.

(46) According to some embodiments, the antibody is directly linked to the toxin. According to other embodiments, the antibody and the toxin are linked through a linker. According to some embodiments, the humanized described herein is covalently linked to the toxin.

(47) According to some embodiments, the linker is cleavable. According to additional embodiments, the linker is not cleavable.

(48) According to some embodiments, the linker is cleaved in response to changes in pH or redox potential. According to some embodiments, the linker is cleaved when contacted with lysosomal enzymes.

(49) The present invention provides, according to another aspect, a pharmaceutical composition comprising the humanized antibody or antigen binding fragment described herein or a conjugate comprising the antibody and a pharmaceutically acceptable excipient, carrier, or diluent.

(50) According to other embodiments, the pharmaceutical composition according to the invention is for use in treating cancer characterized by overexpression of Nectin-2. Nectin-2 overexpression related cancer types can be identified using known data bases such as The Cancer Genome Atlas (TCGA). According to certain embodiments, the cancer treatable with a composition according to the present invention is selected from the group consisting of adrenocortical carcinoma (ACC), chromophobe renal cell carcinoma (KICH), liver hepatocellular carcinoma (LIHC), colon and rectal adenocarcinoma (COAD, READ), pancreatic ductal adenocarcinoma (PAAD), pheochromocytoma & paraganglioma (PCPG), papillary kidney carcinoma (KIRP), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), prostate adenocarcinoma (PRAD), uterine corpus endometrial carcinoma (UCEC), cervical cancer (CESC), cutaneous melanoma (SKCM), mesothelioma (MESO), urothelial bladder cancer (BLCA), clear cell kidney carcinoma (KIRC), lung squamous cell carcinoma (LUSC), uterine carcinosarcoma (UCS), sarcoma (SARC), ovarian serous cystadenocarcinoma (OV), papillary thyroid carcinoma (THCA), glioblastoma multiforme (GBM), breast cancer (BRCA), lower grade glioma (LGG), and diffuse large B-cell lymphoma (DLBC). Each possibility represents a separate embodiment of the invention.

(51) As used herein the term individual, patient, or subject refers to individuals diagnosed with, suspected of being afflicted with, or at-risk of developing at least one disease for which the described compositions and method are useful for treating. According to some embodiments the individual is a mammal. According to some embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. According to some embodiments, the individual is a human.

(52) As used herein the term an effective amount refers to the amount of a therapeutic that causes a biological effect when administered to a mammal. Biological effects include, but are not limited to, inhibition or blockade of a receptor ligand interaction (e.g., PVR-TIGIT, PD-1-PD-L1/PD-L-2), inhibition of a signaling pathway, reduced tumor growth, reduced tumor metastasis, or prolonged survival of an animal bearing a tumor. A therapeutic amount is the concertation of a drug calculated to exert a therapeutic effect. A therapeutic amount encompasses the range of dosages capable of inducing a therapeutic response in a population of individuals. The mammal can be a human individual. The human individual can be afflicted with or suspected or being afflicted with a tumor.

(53) As used herein the term combination or combination treatment can refer either to concurrent administration of the articles to be combined or sequential administration of the articles to be combined. As described herein, when the combination refers to sequential administration of the articles, the articles can be administered in any temporal order.

(54) As used herein checkpoint inhibitor refers a drug that inhibits a biological molecule (checkpoint molecule) produced by an organism that negatively regulates the anti-tumor/cancer activity of T cells in the organism. Checkpoint molecules include without limitation PD-1, PD-L-1, PD-L-2, CTLA4, TIM-3, LAG-3, VISTA, SIGLEC7, PVR, TIGIT, IDO, KIR, A2AR, B7-H3, B7H4, CEACAM1, and CD112R.

(55) The molecules of the present invention as active ingredients are dissolved, dispersed or admixed in an excipient that is pharmaceutically acceptable and compatible with the active ingredient as is well known. Suitable excipients are, for example, water, saline, phosphate buffered saline (PBS), dextrose, glycerol, ethanol, or the like and combinations thereof. Other suitable carriers are well known to those skilled in the art. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents.

(56) The term treatment as used herein refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.

(57) The terms cancer and tumor relate to the physiological condition in mammals characterized by deregulated cell growth. Cancer is a class of diseases in which a group of cells display uncontrolled growth or unwanted growth. Cancer cells can also spread to other locations, which can lead to the formation of metastases. Spreading of cancer cells in the body can, for example, occur via lymph or blood. Uncontrolled growth, intrusion, and metastasis formation are also termed malignant properties of cancers. These malignant properties differentiate cancers from benign tumors, which typically do not invade or metastasize.

(58) According to some embodiments, the method of treating cancer comprises administering the pharmaceutical composition as part of a treatment regimen comprising administration of at least one additional anti-cancer agent.

(59) According to some embodiments, the anti-cancer agent is selected from the group consisting of an antimetabolite, a mitotic inhibitor, a taxane, a topoisomerase inhibitor, a topoisomerase II inhibitor, an asparaginase, an alkylating agent, an antitumor antibiotic, and combinations thereof. Each possibility represents a separate embodiment of the invention.

(60) According to some embodiments, the antimetabolite is selected from the group consisting of cytarabine, fludarabine, fluorouracil, mercaptopurine, methotrexate, thioguanine, gemcitabine, and hydroxyurea. According to some embodiments, the mitotic inhibitor is selected from the group consisting of vincristine, vinblastine, and vinorelbine. According to some embodiments, the topoisomerase inhibitor is selected from the group consisting of topotecan and irinotecan. According to some embodiments, the alkylating agent is selected from the group consisting of busulfan, carmustine, lomustine, chlorambucil, cyclophosphamide, cisplatin, carboplatin, ifosfamide, mechlorethamine, melphalan, thiotepa, dacarbazine, and procarbazine. According to some embodiments, the antitumor antibiotic is selected from the group consisting of bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone, and plicamycin. According to some embodiments, the topoisomerase II is selected from the group consisting of etoposide and teniposide. Each possibility represents a separate embodiment of the present invention.

(61) The present invention provides, according to another aspect, a method of treating a cancer in an individual afflicted with a cancer comprising administering to the individual a therapeutically effective amount of the humanized antibody or antigen binding fragment thereof or the pharmaceutical composition, and an inhibitor of PD-1, PD-L1, CTLA-4 or CD112R signaling. In certain embodiments, the cancer comprises a solid tumor. In certain embodiments, the cancer is selected from the group consisting of lung cancer, colon cancer, glioblastoma, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, cervical cancer, or prostate cancer. In certain embodiments, the inhibitor of PD-1 signaling is an antibody or fragment thereof that binds to PD-1. In certain embodiments, the antibody or fragment thereof that binds to PD-1 is Pembrolizumab, Nivolumab, AMP-514, Tislelizumab, Spartalizumab, or a PD-1 binding fragment thereof. In certain embodiments, the inhibitor of PD-1 signaling is an antibody that specifically binds PD-L-1 or PD-L-2. In certain embodiments, the antibody that specifically binds PD-L1 or PD-L2 comprises Durvalumab, Atezolizumab, Avelumab, BMS-936559, or FAZ053, or a PD-L1 or PD-L2 binding fragment thereof. In certain embodiments, the inhibitor of PD-1 signaling comprises an Fc-fusion protein that binds PD-1, PD-L1, or PD-L2. In certain embodiments, the Fc-fusion protein comprises AMP-224 or a PD-1 binding fragment thereof. In certain embodiments, the inhibitor of PD-1 signaling comprises a small molecule inhibitor of PD-1, PD-L1, or PD-L2. In certain embodiments, the small molecule inhibitor of PD-1, PD-L1, or PD-L2 signaling comprises on or more of: N-{2-[({2-methoxy-6-[(2-methyl[1,1-biphenyl]-3-yl) methoxy]pyridin-3-yl}methyl)amino]ethyl}acetamide (BMS 202); (2-((3-cyanobenzyl)oxy)-4-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylbenzyl)oxy)-5-methylbenzyl)-D-serine hydrochloride; (2R,4R)-1-(5-chloro-2-((3-cyanobenzyl)oxy)-4-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylbenzyl)oxy)benzyl)-4-hydroxypyrrolidine-2-carboxylic acid; 3-(4,6-dichloro-1,3,5-triazin-2-yl)-1-phenylindole; 3-(4,6-dichloro-1,3,5-triazin-2-yl)-1-phenyl-1h-indole; L--Glutamine, N.sup.2,N.sup.6-bis(L-scryl-L-asparaginyl-L-threonyl-L-seryl-L--glutamyl-L-seryl-L-phenylalanyl)-L-lysyl-L-phenylalanyl-L-arginyl-L-valyl-L-threonyl-L-glutaminyl-L-leucyl-L-alanyl-L-prolyl-L-lysyl-L-alanyl-L-glutaminyl-L-isoleucyl-L-lysyl; (2S)-1-[[2,6-dimethoxy-4-[(2-methyl[1,1-biphenyl]-3-yl) methoxy]phenyl]methyl]-2-piperidinecarboxylic acid; Glycinamide, N-(2-mercaptoacetyl)-L-phenylalanyl-N-methyl-L-alanyl-L-asparaginyl-L-prolyl-L-histidyl-L-leucyl-N-methylglycyl-L-tryptophyl-L-seryl-L-tryptophyl-N-methyl-L-norleucyl-N-methyl-L-norleucyl-L-arginyl-L-cysteinyl-, cyclic (1.fwdarw.14)-thiocther; or a derivative or analog thereof.

(62) Also described herein is a method of making composition for treating a cancer in an individual afflicted with cancer comprising admixing the humanized antibody or antigen binding fragment thereof and a pharmaceutically acceptable excipient, carrier, or diluent. In certain embodiments, the cancer comprises a solid tumor. In certain embodiments, the cancer is selected from the group consisting of colon cancer, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, glioblastoma, cervical cancer, prostate cancer, and lung cancer.

(63) Also described herein is a method of producing the humanized antibody or antigen binding fragment thereof comprising incubating the cell line described herein in a cell culture medium under conditions sufficient to allow expression and secretion of the humanized antibody or antigen binding fragment thereof.

(64) According to some particular embodiments, the additional anti-cancer agent is selected from the group consisting of bevacizumab, carboplatin, cyclophosphamide, doxorubicin hydrochloride, gemcitabine hydrochloride, topotecan hydrochloride, thiotepa, and combinations thereof. Each possibility represents a separate embodiment of the present invention.

EXAMPLES

(65) Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non-limiting fashion.

(66) Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, immunological, and recombinant DNA techniques. Such techniques are well known in the art. Other general references referring to well-known procedures are provided throughout this document for the convenience of the reader.

Example 1. High Expression of Nectin-2 mRNA Correlates with Poor Survival Probability of Various Cancer Patients

(67) The correlation between Nectin-2 mRNA expression levels and survival probability was examined on data from TCGA site, and analyzed using the oncolnc.org site, (https://doi.org/10.7717/peerj-cs.67). Nectin-2 mRNA expression levels were the basis for dividing patients for two subgroups of low and high expressors, as indicated by the arrows in FIG. 1 for Low grade glioma (FIG. 1A; p=5.22E-5), Kidney Renal Clear Cell Carcinoma (FIG. 1B; p=0.00037) and lung adenocarcinoma (FIG. 1C; p=0.0319) patients.

Example 2. Nectin-2 Binds and Affects Immune Cells Through Specific Receptors

(68) The schematic illustration of FIG. 2 demonstrates receptors expressed on immune cells and their respective affinities to Nectin-2 expressed by tumors or on antigen presenting cells (APCs). TIGIT relates to a co-inhibitory receptor on immune cells such as T and NK cells; DNAM-1 (also termed CD226) relates to an activating receptor on immune cells (e.g., T cells), and CD112R (also termed PVRIG) relates to a co-inhibitory receptor on lymphoid immune cells (e.g., T and NK cells); Nectin-2 (CD112) servs as an inhibitory ligand for immune cells, mainly via its binding to CD112R. According to the present invention, humanized anti-Nectin-2 mAbs block Nectin-2 interactions with its high affinity receptor CD112R and may target cancer cells for specific toxin delivery and killing.

Example 3. Nectin-2 is Expressed on the Majority of Solid Tumors

(69) The database Proteinatlas.com was searched for all the aliases of Nectin-2 (NECTIN2, CD112, HVEB, PRR2, PVRL2, PVRR2). Under the pathology rubric, data using three different mAbs was found. HPA0127569 mAb has the highest validation score (Enhanced) as it was validated by orthogonal method. Thus, the expression data across different tumors was selected for this clone only, and is depicted in FIG. 3. The graph is showing percent of tumors positive for Nectin-2 expression. In 17/20 indications membranous expression of Nectin-2 is seen at Moderate-High levels.

Example 4. Improved Properties of Humanized Anti-Nectin-2 mAbs

(70) Murine anti-human Nectin-2 clone 2.11, disclosed in Patent application publication No. WO2020144697, was selected as the lead mAb for humanization. Based on structural analysis, a large preliminary set of sequence segments were identified that were used to create the humanized variants. These segments were selected and analyzed using iTope technology for in silico analysis of peptide binding to human MHC class II alleles (Perry et al., 2008) and using the TCED of known antibody sequence-related T cell epitopes (Bryson et al., 2010). Sequence segments that were identified as significant non-human germline binders to human MHC class II, or that scored significant hits against the TCED, were discarded. This resulted in a reduced set of segments, and combinations of these were further analyzed, as described above, to ensure that the junctions between segments did not contain potential T cell epitopes. Selected sequence segments were assembled into complete V region sequences that were devoid of significant T cell epitopes. Five heavy chain (VH1 to VH5) and 5 light chain (VK1 to VK5) sequences were then chosen. The data at Table 1 present the readout of the iTope algorithm. To reduce the potential for anti-drug antibody (ADA) generation, the sequence of the mAbs was analyzed for potential binding of MHCI1. The main risk is linked to the high affinity binding peptides, which are separated from the moderated affinity binding peptides.

(71) TABLE-US-00001 TABLE 1 Improved characteristics of the humanized anti-Nectin-2 mAbs. Illustration of predicted MHCII epitopes (iTope score) of the parental (V0) and humanized heavy (VH1-5) and light (Vk1-5) variable chains used to generate humanized variants for lead drug selection. Heavy Chain Predicted affinity MHCII VH0 VH1 VH2 VH3 VH4 VH5 Moderate 5 6 4 4 4 3 High 8 4 3 3 1 1 Light Chain Predicted affinity MHCII Vk0 Vk1 Vk2 Vk3 Vk4 Vk5 Moderate 8 4 3 3 3 1 High 4 3 3 2 2 2

(72) The parental heavy chain (VH0) has 8 predicted high affinity motifs and the parental light chain (Vk0) has 4 such motifs. Following the humanization process, the number of predicted high affinity MHCII epitopes for the heavy chain was reduced to 3 (VH2, VH3) or even to 1 (VH4, VH5) and for the light chain the predicted high affinity MHCII motifs were reduced to 2 (VK3-5).

(73) Thus, the humanization process eliminated the majority of the predicted high affinity MHCII motifs that may trigger immunogenicity against the mAb.

(74) Stability of the humanized variants was assessed by thermal ramp stability experiments that are well established methods for ranking proteins and formulations for stability. A protein's denaturation profile provides information about its thermal stability and represents a structural fingerprint for assessing structural and formulation buffer modifications. A widely used measure of the thermal structural stability of a protein is the temperature at which it unfolds from the native state to a denatured state.

(75) For many proteins, this unfolding process occurs over a narrow temperature range and the mid-point of this transition is termed melting temperature or Tm. To determine the melting temperature of a protein, UNcle measures the fluorescence of Sypro Orange (which binds to exposed hydrophobic regions of proteins) as the protein undergoes conformational changes. Increased Tm is a desired characteristic in Ab lead selection as it predicts a more stable Ab.

(76) Purified lead humanized antibody variants, in duplicates, were diluted to a final test concentration of 0.5 mg/ml in PBS into which Sypro Orange (at 160 Stock solution) was added to a final concentration of 20 solution. 9 L of each sample mixture was loaded in duplicate into UNi microcuvettes. Samples were subjected to a thermal ramp from 15-95 C., with a ramp rate of 0.3 C./minute and excitation at 473 nm. Full emission spectra were collected from 250-720 nm, and the area under the curve between 510-680 nm was used to calculate the inflection points of the transition curves (Tonset and Tm). As seen in Table 2, for all of the antibodies tested, Tm1 and Tonset are higher for the humanized variants compared with the chimeric antibody (VH0/VK0).

(77) TABLE-US-00002 TABLE 2 A summary of thermal stability values for the parental/chimeric (VH0/Vk0) antibody and for six purified lead humanized variants as determined using the UNcle biostability platform. Variant Average Tonset ( C.) Average Tm1 ( C.) VH0/V0 58.1 67.3 VH3/V3 65.6 74.3 VH4/V2 60.7 71.9 VH4/V3 60.6 70.8 VH5/V3 58.8 72.7 VH5/V4 59.9 68.3 VH5/V5 61.3 69.4 Improved stability of both Tonset and TM1 (measuring of unfolding) above 5 degrees Celsius, was considered significant and is marked in bold fonts.

Example 5. Improved Binding to Nectin-2, and Blocking of CD112R Binding by the Humanized Anti-Nectin-2 mAbs

(78) The humanized anti-Nectin-2 mAbs binding to human Nectin-2 expressed by 293T cells, (protein id: Q92692) and Chlorocebus (African green monkey, AFG) Vero cells were assessed and EC50 values were established. AFG expresses Nectin-2 protein (XP_007995342.1) with 97% similarity to human Nectin-2. FIGS. 4A and 4B depict fold change of EC50 values to human and AFG cells expressed Nectin-2, respectively. Both cell lines were plated at 0.510.sup.5 cells per well. The chimeric (HOKO) and humanized mAbs were added at concentration range of 20-0.001 nM. For detection, anti-human-APC Ab was used at 1:200 dilution (Jackson Immunoresearch AB_2340526). Cells were analyzed by FACS and EC50 was calculated and compared to that of the chimeric Ab (fold improve). This analysis revealed improved binding of the humanized variants, and similar degree of cross-reactivity to monkey Nectin-2, comparing to the chimeric mAb (FIGS. 4A vs. 4B). To evaluate the blocking capacity of the humanized Abs, human Nectin-2 was overexpressed in Chinese hamster ovary (CHO) cells. FIG. 4C shows data generated when 10.sup.5 of CHO-hNectin-2/well were incubated with 30 nM of human CD112R-mlgG2a in presence of the indicated humanized Abs, in concentrations ranging from 66-0.81 nM. Bound CD112R was detected by using amlgG2a-647 (Jackson Immunoresearch Cat 115-607-186) at 1:200 dilution followed by FACS analysis. As can be seen, all humanized variants maintained full blocking capacity.

Example 6. Anti-hNectin-2 Humanized Variant H3K3 can Serve as an ADC Driver in an Fc Independent Manner

(79) To assess the capacity of the mAbs to serve as ADCs, the streptavidin-saporin (ZAP), IT-27-250 (ATS), was used. The mAbs indicated in FIG. 5 were biotinylated using the biotinylation kit Ab207195 (Abcam) at 1:1 ratio. FIG. 5A depicts the results of the initial screen which included top humanized clones and A549 (lung adenocarcinoma) cells as targets. 210.sup.3 cells per well were plated and allowed to adhere over 4-6 hours period. The ADCs were added, and the cells were incubated with the ADCs. After 72 hours, the assay was harvested and tumor cell killing was evaluated using CellTiter-Glo 2.0 Cell Viability Assay-(Promega G9242) following standard protocol. Only clone H3K3 showed robust killing. Next, clone H3K3 was directly compared to clone H4K2 (FIG. 5B). Both clones exhibited robust killing, and H3K3 was selected for future analysis due to superior developability properties. The chimeric parental clone and the FcgR.sup.null variant (IgG2-P238H) (In FcgR.sup.null, the g is for gamma or ) of clone H3K3 (IgG1) were tested under the above conditions and results are shown in FIG. 5C. Significant killing (p<0.001) by both the chimeric and humanized H3K3 variants was seen at all concentrations of these ADCs, and the FcgR.sup.null variant led to identical level of target cell killing compared to the hlgG1 clone.

Example 7. Anti-hNectin-2 H3K3-FcgR.SUP.null .ADCs Lead to Robust Killing of Solid Tumor Cell Lines In-Vitro

(80) Additional H3K3-FcgR.sup.null mAbs were generated by mutating key residues in the hinge region of human IgG1 as indicated in Table 3.

(81) TABLE-US-00003 TABLE 3 A summary of the Fc-substitution for the humanized IgG1 FcgR.sup.null linker-payload combinations, release mechanisms, and the relevant drug-to-antibody ratios (DARs) for the H3K3-based ADCs. Average Fc-Mutation Release DAR (FcgR.sup.null) Linker Payload mechanism (LC-MS) hIgG1 G237I MC-VC-PAB MMAE Proteolytic 4.3 cleavage hIgG1 G237I MC MMAF Degradation 4.2 hIgG1 L235S SMCC DM1 Degradation 4.0 hIgG1 L235S SPDB DM4 Redox 4.2 hIgG1 L235S/G237I Lys-PAB-CO SN38 pH 8

(82) The presented linker payload combinations were chosen according to the desired release mechanism and were generated according to the standard protocol by Abzena LTD. Briefly, the mAbs were reduced and incubated with an excess of the linker-payload to obtain the desired drug antibody ratio (DAR) of 4 for all the linker-payloads except for SN-38, which had a target DAR of 8. The final products were then purified, and the DARs were established by LC/MS method. Selected tumor cell lines, representing various solid tumors, were used to assess potency of the various linker-payload combinations in-vitro. The indicated target cells were plated at 210.sup.3 cells per well and allowed to adhere over 4-6 hours period. The ADCs were added at concentrations of 4-0.16 ug/ml using 5-fold dilutions, and the cells were incubated with the ADCs. After 72 hours, the assay was harvested and tumor cell killing was evaluated using CellTiter-Glo 2.0 Cell Viability Assay(Promega G9242) following standard protocol. Robust killing of RKO cells (colorectal adenocarcinoma), MDA-MB-231 cells (triple negative breast cancer) and of A549 cells (lung adenocarcinoma) is depicted in FIGS. 6A-6C, respectively. Killing of target cells was significant (p<0.001) by two-tailed student t-test, unless indicated by NS (not significant, p>0.05).

Example 8. Selected FcgR.SUP.null .Mutations Prevent ADC Killing Via FcgR

(83) To evaluate the degree of non-target specific killing by the humanized anti-Nectin-2 ADCs, which is due to the Ab Fc binding to its FcR, and which may contribute to non-specific effect of the ADC, CHO cells overexpressing the high affinity Fc receptor hCD64 (CHO-hCD64) were generated. These cells do not express human Nectin-2 and thus no target-specific killing is expected. Parental CHO cells and CHO-hCD64 were plated at 210.sup.3 cells per well and allowed to adhere over 4-6 hours. The different Fc variant ADCs were added at 12 ug/ml. After incubation of 72 hours, the assay was harvested and tumor cell killing was evaluated using CellTiter-Glo 2.0 Cell Viability Assay-(Promega G9242).

(84) As seen in FIG. 7, both the IgG4-S228P and the hlgG1-positive controls, led, as expected, to significantly higher degree of the killing of the CHO-hCD64 cells compared to parental CHO cells. On the other hand, the IgG1 G237I substitution resulted in similar low-level of killing of both parental and hCD64 overexpressing cells (non-specific killing). These results confirm that the FogRnull variant is not bound and internalized by the high affinity FcgR CD64.

Example 9. Anti-hNectin-2 Humanized H3K3-FcgR.SUP.null .ADCs Lead to Robust Killing of the Hematological Tumor Cell Line HL-60 In-Vitro

(85) HL-60 cells (AML model) were plated at 210.sup.3 cells per well and the ADCs were added at concentrations of 9-1 g/ml, using 3-fold dilutions. The cells were incubated with the ADCs for 72 hours. Then, the assay was harvested and tumor cell killing was evaluated using CellTiter-Glo 2.0 Cell Viability Assay(Promega G9242) as described above. As seen in FIG. 8, except for the DM1 payload (indicated in the graph), all other compounds led to significant killing of the targets in all concentrations tested and all exceeded 90% killing at the highest concentration (>60 nM). Based on these results the SMCC-DM1 linker payload was excluded from future experiments.

Example 10. Anti-hNectin-2 H3K3-FcgR.SUP.null.-MC-VC-PAB-MMAE ADC Leads to Tumor Regression in an Aggressive AML Model

(86) Nude female mice (n=25) were injected s.c. with 1010.sup.6 HL-60 cells in 1:1 Matrigel. Once tumors reached an average volume of 205 mm.sup.3, mice were randomized into five groups (n=5 per group) and treated in a blinded manner, by i.v. injection of either PBS (vehicle), H3K3-FcgR.sup.null_MMAE, H3K3-FcgR.sup.null-MMAF, H3K3-FcgR.sup.null-DM4 or H3K3-FcgR.sup.null-SN-38. All treatments were at 5 mg/kg, given every 4 days, for 4 consecutive doses. As can be seen in FIG. 9A, on day 23 after randomization, H3K3-FcgR.sup.null-MMAF and the SN-38 variants did not attenuate tumor growth. H3K3-FcgR.sup.null-DM4 led to significant tumor growth inhibition (TGI) of 49%, while H3K3-FcgR.sup.null-MMAE led to significant tumor regression. FIG. 9B shows the mean effect of H3K3-FcgR.sup.null-MMAE.

Example 11. Anti-hNectin-2 H3K3-FcgR.SUP.null.-MC-VC-PAB-MMAE ADC Leads to Tumor Regression in an Aggressive Colon Adenocarcinoma Model

(87) Nude female mice (n=5 per group) were injected SC with 510.sup.6 RKO cells in 1:1 Matrigel. Once tumors reached an average volume of 160 mm.sup.3, mice were randomized into two groups and treated every 4 days, in a blinded manner, by i.v. injection of either PBS (Vehicle) or H3K3-FcgR.sup.null-MMAE (1107-MC-VC-MMAE) at 5 mg/kg for 4 consecutive doses. On day 13 after randomization, H3K3-FcgR.sup.null-MMAE, led to significant tumor regression, as can be seen in FIG. 10, representing >80% TGI compared to vehicle treated group.

Example 12. Anti-hNectin-2 H3K3-FcgR.SUP.null .mAb has Similar In Vitro Killing Activity Compared to Trodelvy

(88) For the in vitro killing assay (FIG. 11) MDA-MB-231 target cells were plated at 210.sup.3 cells per well and allowed to adhere over 4-6 hours. Next, H3K3-FcgR.sup.null anti-Nectin-2 mAb (NTX1107) linked to Dxd, and Trodelvy (Sacituzumab govitecan), an approved ADC drug for TNBC, were added at concentrations of 12 ug/ml (dark grey bars) and 3 ug/ml (light grey bars). Of note, both ADCs use the same class of cytotoxic payload (i.e., topoisomerase I (TOP1) inhibitors). The cells were incubated with the ADCs for 120 hours, after which tumor cell killing was evaluated using CellTiter-Glo 2.0 Cell Viability Assay-(Promega G9242), following a standard protocol. Robust killing of MDA-MB-231 cells was induced by both ADCs with significant superiority of Trodelvy at both concentrations (*** p<0.0005).

Example 13. Anti-hNectin-2 H3K3-FcgR.SUP.null .Conjugated to Topoisomerase 1 Inhibitor, but not Trodelvy, Inhibits In Vivo Growth of Aggressive MDA-MB-231 Tumors

(89) Nude female mice (n=7 per group) were injected s.c. with 510.sup.6 MDA-MB-231 cells in 1:1 Matrigel. Once tumors reached an average volume of 230 mm.sup.3 mice were randomized into treatment groups and treated every 4 days, for 3 consecutive doses, in a blinded manner, by i.v. injection of either PBS (vehicle), NTX1107-Dxd, or Trodelvy at 5 mg/kg. There was no effect for Trodelvy treatment on the tumor growth.

(90) As seen in FIG. 12, NTX1107-Dxd was able to significantly inhibit tumor growth (*p<0.02, **<0.005.*** p<0.0005). These findings were unexpected since the in-vitro killing potency of Trodelvy was higher than that of NTX1107-Dxd FIG. 11), and Trodelvy is an approved ADC for TNBC. These observations suggest that anti-Nectin-2 (NTX1107) is a uniquely potent ADC for the treatment of solid tumors.

Example 14. Anti-hNectin-2 H3K3-FcgR.SUP.null .can Regress Aggressive MDA-MB-231 Tumors In Vivo when Conjugated to Tubulin or TOP1 Targeting Agents

(91) Nude female mice (n=7 per group) were injected s.c. with 510.sup.6 MDA-MB-231 cells in 1:1 Matrigel. Once tumors reached an average volume of 210 mm.sup.3 mice were randomized into treatment groups and treated every 4 days, for 5 consecutive doses, in a blinded manner, by i.v. injection of either PBS (vehicle), H3K3-FcgR.sup.null (NTX1107)-MMAE, or NTX1107-Dxd. As can be seen in FIG. 13, all animals treated with NTX1107-MMAE had no detectable tumors at the end of the study. For the animals treated with NTX1107-Dxd, four had no tumors, while for the remaining three the average tumor volume was 210 mm.sup.3, indicating tumor stasis. These findings suggest that the humanized antibodies targeting Nectin-2 described herein (NTX1107) may form uniquely potent ADCs for treatment of solid tumors, when using payloads of various classes.

(92) Sequences

(93) TABLE-US-00004 TABLE4 CDRsequences: Overlapping Description KABAT IMGT sequence HeavyChain SYWIH GYIFTSYW SYW CDR1 (SEQIDNO:1) (SEQIDNO:7) HeavyChain AVYPGNSDSNYNQKF(KA/QG) VYPGNSDS SEQID CDR2 (SEQIDNO:2) (SEQIDNO:8) NO:8 HeavyChain LVGTFDY TKLVGTFDY SEQID CDR3 (SEQIDNO:3) (SEQIDNO:9) NO:3 LightChain (K/R)ASQNVGINV(V/A) QNVGIN SEQID CDR1 (SEQIDNO:4) (SEQIDNO:10) NO:10 LightChain SASYRYS SAS SAS CDR2 (SEQIDNO:5) LightChain QQYNTNPFT SEQIDNO:6 SEQID CDR3 (SEQIDNO:6) NO:6

(94) TABLE-US-00005 -heavychainvariableregionNT20-11_VH3 SEQIDNO:11 QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYWIHWVRQPPGKGLEWIG AVYPGNSDSNYNQKFKARVTITAVTSTSTAYMELSSLRSEDTAVYYCTK LVGTFDYWGQGTTVTVSS lightchainvariableregionNT20-11_Vk3 SEQIDNO:12 DIQMTQSPSTLSASVGDRVSVTCKASQNVGINVVWYQQKPGQPPKTLIY SASYRYSGVPDRFSGSGSGTDFTLTISSLQAEDLAEYFCQQYNTNPFTF GQGTKLEIK -heavychainvariableregionNT20-11_VH1 SEQIDNO:13 EVQLVQSGTELKKPGSSVKVSCKASGYIFTSYWIHWVRQPPGKGLEWIG AVYPGNSDSNYNQKFKARATITAVTSTSTAYMELSSLTSEDSAVYYCTK LVGTFDYWGQGTTVTVSS -heavychainvariableregionNT20-11_VH2 SEQIDNO:14 EVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYWIHWVRQPPGKGLEWIG AVYPGNSDSNYNQKFKARATITAVTSTSTAYMELSSLRSEDTAVYYCTK LVGTFDYWGQGTTVTVSS -heavychainvariableregionNT20-11_VH4 SEQIDNO:15 QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYWIHWVRQPPGKGLEWIG AVYPGNSDSNYNQKFQGRVTITAVTSTSTAYMELSSLRSEDTAVYYCTK LVGTFDYWGQGTTVTVSS -heavychainvariableregionNT20-11_VH5 SEQIDNO:16 QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYWIHWVRQPPGKGLEWIG AVYPGNSDSNYNQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTK LVGTFDYWGQGTTVTVSS -lightchainvariableregionNT20-11_Vk1 SEQIDNO:17 DIVMTQSPSFLSASVGDRVSVTCKASQNVGINVVWYQQRAGQPPKTLIY SASYRYSGVPDRFTGSGSGTDFTLTISSLQSEDLAEYFCQQYNTNPFTF GQGTKLEIK -lightchainvariableregionNT20-11_Vk2 SEQIDNO:18 DIVMTQSPSTLSASVGDRVSVTCKASQNVGINVVWYQQKPGQPPKTLIY SASYRYSGVPDRFTGSGSGTDFTLTISSLQAEDLAEYFCQQYNTNPFTF GQGTKLEIK -lightchainvariableregionNT20-11_Vk4 SEQIDNO:19 DIQMTQSPSTLSASVGDRVTITCRASQNVGINVVWYQQKPGQPPKTLIY SASYRYSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYNTNPFTF GQGTKLEIK -lightchainvariableregionNT20-11_Vk5 SEQIDNO:20 DIQMTQSPSTLSASVGDRVTITCRASQNVGINVAWYQQKPGQPPKTLIY SASYRYSGVPDRESGSGSGTDFTLTISSLQAEDVAVYYCQQYNTNPFTF GQGTKLEIK

(95) TABLE-US-00006 TABLE5 Framework(Non-CDR)sequencesofthehumanizedheavychainvariableregions. Chain FR-H1 FR-H2 FR-H3 FR-H4 VH3 QVQLVQSGAEVKKPGSSV WVRQPPGKGLEWI RVTITAVTSTSTAYM WGQGTTVTV KVSCKASGYIFT(SEQID G(SEQIDNO:22) ELSSLRSEDTAVYYC SS(SEQID NO:21) TK(SEQIDNO:23) NO:24) VH1 EVQLVQSGTELKKPGSSV WVRQPPGKGLEWI RATITAVTSTSTAYM WGQGTTVTV KVSCKASGYIFT(SEQID G(SEQIDNO:22) ELSSLTSEDSAVYYCT SS(SEQID NO:25) K(SEQIDNO:26) NO:24) VH2 EVQLVQSGAEVKKPGSSV WVRQPPGKGLEWI RATITAVTSTSTAYM WGQGTTVTV KVSCKASGYIFT(SEQID G(SEQIDNO:22) ELSSLRSEDTAVYYC SS(SEQID NO:27) TK(SEQIDNO:28) NO:24) VH4 QVQLVQSGAEVKKPGSSV WVRQPPGKGLEWI RVTITAVTSTSTAYM WGQGTTVTV KVSCKASGYIFT(SEQID G(SEQIDNO:22) ELSSLRSEDTAVYYC SS(SEQID NO:21) TK(SEQIDNO:23) NO:24) VH5 QVQLVQSGAEVKKPGSSV WVRQPPGKGLEWI RVTITADESTSTAYM WGQGTTVTV KVSCKASGYIFT(SEQID G(SEQIDNO:22) ELSSLRSEDTAVYYC SS(SEQID NO:21) TK(SEQIDNO:29) NO:24)

(96) TABLE-US-00007 TABLE6 Framework(Non-CDR)sequencesofthehumanizedlightvariableregions. Chain FR-L1 FR-L2 FR-L3 FR-L4 LK3 DIQMTQSPSTLSASVGDR WYQQKPGQPPKTLIY GVPDRFSGSGSGTDF FGQGTKLEI VSVTC(SEQIDNO:30) (SEQIDNO:31) TLTISSLQAEDLAEYF K(SEQID C(SEQIDNO:32) NO:33) LK1 DIVMTQSPSFLSASVGDR WYQQRAGQPPKTLIY GVPDRFTGSGSGTDF FGQGTKLEI VSVTC(SEQIDNO:34) (SEQIDNO:35) TLTISSLQSEDLAEYF K(SEQID C(SEQIDNO:36) NO:33) LK2 DIVMTQSPSTLSASVGDR WYQQKPGQPPKTLIY GVPDRFTGSGSGTDF FGQGTKLEI VSVTC(SEQIDNO:37) (SEQIDNO:31) TLTISSLQAEDLAEYF K(SEQID C(SEQIDNO:38) NO:33) LK4 DIQMTQSPSTLSASVGDR WYQQKPGQPPKTLIY GVPDRFSGSGSGTDF FGQGTKLEI VTITC(SEQIDNO:39) (SEQIDNO:31) TLTISSLQAEDVAVYY K(SEQID C(SEQIDNO:40) NO:33) LK5 DIQMTQSPSTLSASVGDR WYQQKPGQPPKTLIY GVPDRFSGSGSGTDF FGQGTKLEI VTITC(SEQIDNO:39) (SEQIDNO:31) TLTISSLQAEDVAVYY K(SEQID C(SEQIDNO:40) NO:33)

(97) TABLE-US-00008 -HCCDR2 SEQIDNO:41 AVYPGNSDSNYNQKFKA -HCCDR2 SEQIDNO:42 AVYPGNSDSNYNQKFQG -LCCDR1 SEQIDNO:43 KASQNVGINVV -LCCDR1 SEQIDNO:44 KASQNVGINVA -LCCDR1 SEQIDNO:45 RASQNVGINVV -LCCDR1 SEQIDNO:46 RASQNVGINVA -aminoacidsequenceofVH3FulllengthhlgG1heavychain SEQIDNO:47 QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYWIHWVRQPPGKGLEWIGAVYPGNS DSNYNQKFKARVTITAVTSTSTAYMELSSLRSEDTAVYYCTKLVGTFDYWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK -DNASequenceVH3FulllengthhIgG1 SEQIDNO:48 caggtgcagctggtgcagagcggcgcggaagtgaaaaaaccgggcagcagcgtgaaagtg agctgcaaagcgagcggctatatttttaccagctattggattcattgggtgcgccagccg ccgggcaaaggcctggaatggattggcgcggtgtatccgggcaacagcgatagcaactat aaccagaaatttaaagcgcgcgtgaccattaccgcggtgaccagcaccagcaccgcgtat atggaactgagcagcctgcgcagcgaagataccgcggtgtattattgcaccaaactggtg ggcacctttgattattggggccagggcaccaccgtgaccgtgagcagcgcgagcaccaaa ggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcg ctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggc gcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagc ctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaac gtgaaccataaaccgagcaacaccaaagtggataaacgcgtggaaccgaaaagctgcgat aaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgttt ctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgc gtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggc gtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgc gtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgc aaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggc cagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgaagaaatgaccaaaaac caggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgg gaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgat ggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaac gtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctg agcctgagcccgggcaaa -aminoacidsequenceofVk3FulllengthKappalightchain SEQIDNO:49 DIQMTQSPSTLSASVGDRVSVTCKASQNVGINVVWYQQKPGQPPKTLIYSASYRYSG VPDRESGSGSGTDFTLTISSLQAEDLAEYFCQQYNTNPFTFGQGTKLEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC -DNASequenceVk3FulllengthKappa SEQIDNO:50 gatattcagatgacccagagcccgagcaccctgagcgcgagcgtgggcgatcgcgtgagc gtgacctgcaaagcgagccagaacgtgggcattaacgtggtgtggtatcagcagaaaccg ggccagccgccgaaaaccctgatttatagcgcgagctatcgctatagcggcgtgccggat cgctttagcggcagcggcagcggcaccgattttaccctgaccattagcagcctgcaggcg gaagatctggcggaatatttttgccagcagtataacaccaacccgtttacctttggccag ggcaccaaactggaaattaaacgcaccgtggcggcgccgagcgtgtttatttttccgccg agcgatgaacagctgaaaagcggcaccgcgagcgtggtgtgcctgctgaacaacttttat ccgcgcgaagcgaaagtgcagtggaaagtggataacgcgctgcagagcggcaacagccag gaaagcgtgaccgaacaggatagcaaagatagcacctatagcctgagcagcaccctgacc ctgagcaaagcggattatgaaaaacataaagtgtatgcgtgcgaagtgacccatcagggc ctgagcagcccggtgaccaaaagctttaaccgcggcgaatgc SEQIDNO:51-VH3nucleicacids SEQIDNO:52-VH1nucleicacids SEQIDNO:53-VH2nucleicacids SEQIDNO:54-VH4nucleicacids SEQIDNO:55-VH5nucleicacids SEQIDNO:56-Vk3nucleicacids SEQIDNO:57-Vk1nucleicacids SEQIDNO:58-Vk2nucleicacids SEQIDNO:59-Vk4nucleicacids SEQIDNO:60-Vk5nucleicacids SEQIDNO:61-HumanIgG1(L235S)(includingVH3) SEQIDNO:62-HumanIgG1(L235S/E272K)(includingVH3) SEQIDNO:63-HumanIgG1(G237I)(includingVH3) SEQIDNO:64-HumanIgG1(G237I/E272I)(includingVH3) SEQIDNO:65-HumanIgG1(G237I/V264R)(includingVH3) SEQIDNO:66-HumanIgG1(V215A/E269R/K322A)(includingVH3) SEQIDNO:67-HumanIgG1(L234A/L235A/P329G)(includingVH3) SEQIDNO:68-HumanIgG4(S228P/L235P/V264R)(includingVH3) SEQIDNO:69-HumanIgG2(P238H)(includingVH3) SEQIDNO:70-HumanIgG2(P238H/V264R)(includingVH3) SEQIDNO:71-Leaderpeptidesequence.