HK1246312B - Anti-ceacam6 antibodies and uses thereof - Google Patents

Description

Anti-CEACAM6 antibodies and uses thereof

The present invention provides recombinant antigen-binding regions and antibodies, as well as functional fragments comprising such antigen-binding regions, which are specific for human and cynomolgus monkey (Macaca fascicularis) CEACAM6 (carcinoembryonic antigen-related cell adhesion molecule 6, CD66c, non-specific cross-reacting antigen, NCA, NCA-50/90), and thus do not significantly cross-react with the closely related human CEACAM1, human CEACAM3, and human CEACAM5. The present invention further provides methods for producing such antibodies.

Therefore, the antibodies can be used to treat cancers and other disorders and conditions associated with the expression of CEACAM6. The present invention also provides nucleic acid sequences encoding the aforementioned antibodies, vectors containing the nucleic acid sequences, pharmaceutical compositions, and kits with instructions for use.

Background of the Invention

Antibody-based therapies are effective and clinically established treatments for various cancers (including solid tumors). For example, HERCEPTIN® has been successfully used to treat breast cancer, and RITUXAN® is effective in B-cell related cancer types. The key to developing novel, successful antibody-based therapies is to isolate antibodies directed against cell-surface proteins that are found to be preferentially expressed on target cells (e.g., cancer cells, immune cells, etc.) that are able to functionally alter the activity of the corresponding receptors.

Antibody blocking of immune checkpoint molecules for immune cell activation and therefore for cancer immunotherapy is a clinically validated method. In 2011, the CTLA-4 blocking antibody Ipilimumab was approved by the FDA for 2-line treatment of metastatic melanoma (Yervoy). Another example is blocking the PD-1/PD-L1 axis, for which several drugs have been approved or are currently in clinical development and have reported impressive clinical responses in melanoma, RCC and lung cancer (Henick et al., Expert Opin Ther Targets. 2014 Dec; 18(12): 1407-20).

Proteins of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family belong to the immunoglobulin (Ig) supergene family and generally exhibit a variable (V)-like domain identified as the N domain. The N domain is followed by none or up to six constant C2-like Ig domains (referred to as A or B). These extracellular domains are required for CEACAM functionality as homophilic and heterophilic cell adhesion molecules (Obrinck, Curr Opin Cell Biol. 1997 Oct; 9(5): 616-26) or as receptors for human and rodent pathogens (Kuespert et al., Curr Opin Cell Biol. 2006 Oct; 18(5): 565-71; Voges et al., PLoS One. 2012; 7(6): e39908). CEACAM receptors associate into dimers or oligomers and multiplex with other partners at the cell membrane and thus regulate important functions. In addition to expression in human tissues, the CEACAM gene family is highly conserved among 27 other mammalian species and is best described in mice, rats, cows, dogs, platypus, and opossums (Kammerer and Zimmermann, BMC Biol. 2010 Feb 4;8:12). The best characterized biological function of CEACAM is to support cell-cell adhesion through its homophilic and heterophilic interactions, including roles in differentiation and formation of three-dimensional tissue structures, angiogenesis, apoptosis, tumor suppression, and metastasis (Kuespert et al., Curr Opin Cell Biol. 2006 Oct;18(5):565-71). More details about family members are described in other reviews (Horst and Wagener, Handb Exp Pharmacol. 2004;(165):283-341; Gray-Owen and Blumberg, Nat Rev Immunol. 2006 Jun;6(6):433-46).

CEACAM6 (carcinoembryonic antigen-related cell adhesion molecule 6, CD66c, nonspecific cross-reactive antigen, NCA, NCA-50/90) is a glycosylphosphatidylinositol (GPI)-linked cell surface protein with an N-domain and two C2-like domains that mediates many possible cis- or trans-directed CEACAM interactions through its extracellular domain with various membrane receptors, some of which have been identified (Beauchemin and Arabzadeh, Cancer Metastasis Rev. 2013 Dec;32(3-4):643-71).

CEACAM6 is expressed in various normal human tissues, such as the epithelium of the colon (Blumenthal et al., BMC Cancer, 2007, Jan 3;7:2.), lung (Kolla et al., Am J Physiol Lung Cell Mol Physiol 296:L1019-L1030), and granulocytes (Kuroki et al., Biochem Biophys Res Commun. 1992 Jan 31;182(2):501-6). In the granulocyte lineage, CEACAM6 is expressed at all stages of granulocyte maturation, except in early lineage-specified precursors (Strickland et al., J Pathol. 2009 Jul;218(3):380-90; Schölzel et al., American Journal of Pathology, 156(2), 595-605). CEACAM6 is not expressed in rodents (Beauchemin et al., Exp Cell Res. 1999 Nov 1;252(2):243-9).

CEACAM6 expression has been described for several cancers. In colon cancer, CEACAM6 was upregulated in 55% of cases and was an independent prognostic factor that allowed patients to be subdivided into low-risk and high-risk groups (Jantscheff et al., J Clin Oncol. 2003 Oct 1; 21(19): 3638-46). In pancreatic cancer, 92% (n=82) of the analyzed samples were found to be positive, while CEACAM6 expression was more prevalent in high-grade PanIN lesions than in low-grade PanIN lesions (Duxbury et al., Ann Surg. 2005 Mar; 241(3): 491-6). This was confirmed in another study, in which >90% of invasive pancreatic cancers (110 of 115 tested) showed strong (over) expression of CEACAM6 (Strickland et al., J Pathol. 2009 Jul; 218(3): 380-90). Furthermore, Blumenthal et al. reported CEACAM6 expression in breast tumors, pancreatic tumors, ovarian adenocarcinomas, lung adenocarcinomas, lymph node metastases, and metastases from breast, colon, and lung tumors (Blumenthal et al., BMC Cancer. 2007 Jan 3;7:2).

Others have also reported CEACAM6 expression in breast cancer (Maraqa et al., Clin Cancer Res. 2008 Jan 15;14(2):405-11; Poola et al., Clin Cancer Res. 2006 Aug 1;12(15):4773-83; Balk-Moller et al., Am J Pathol. 2014 Apr;184(4):1198-208); Tsang et al., Breast Cancer Res Treat. 2013 Nov;142(2):311-22). Furthermore, CEACAM6 expression has been reported in multiple myeloma (Witzens-Harig et al., Blood 2013 May 30;121(22):4493-503), gastric cancer (Deng et al., Genet Mol Res. 2014 Sep 26;13(3):7686-97), and head and neck cancer (Cameron et al., Mol Cancer. 2012 Sep 28;11:74).

Experimental evidence supports the role of CEACAM6 as an important regulator of metastasis. Kim et al. have shown that attenuating CEACAM6 expression in LoVo cells or increasing its expression in HCT116 cells using CEACAM6-specific siRNA, respectively, impedes or enhances invasiveness through the extracellular matrix (Kim et al., Clin Chim Acta. 2013 Jan 16;415:12-9). Inhibition of CEACAM6 expression leads to increased E-cadherin promoter activity. Blumenthal et al. have shown that CEACAM5 and CEACAM6 contribute to the metastatic spread of CRC, which can be blocked in vivo by monoclonal antibodies (Blumenthal et al., BMC Cancer. 2007 Jan 3;7:2). It has also been shown that CEACAM6 is expressed in CD133-positive cells in colon cancer samples that are capable of forming stem cell-rich colonospheres, whose proliferation, clonogenic potential, and in vivo tumorigenic potential were significantly impaired upon silencing (Gemei et al., Cancer. 2013 Feb 15;119(4):729-38). In breast cancer, tamoxifen-resistant samples were shown to overexpress CEACAM6 and CEACAM6 was a significant predictor of disease recurrence (Maraqa et al., Clin Cancer Res. 2008 Jan 15;14(2):405-11). In MMU1-tamoxifen-resistant MCF7 cell derivatives, siRNA-mediated silencing of CEACAM6 reversed the endocrine resistance, anchorage-independence, and invasive properties of these cells (Lewis-Wambi et al., Eur J Cancer. 2008 Aug;44(12):1770-9). In lung adenocarcinoma, CEACAM6 expression is significantly associated with poor clinical outcome (Kobayashi et al., Br J Cancer. 2012 Nov 6; 107(10): 1745-53). In pancreatic cancer, silencing CEACAM6 with siRNA reverses the acquired anoikis resistance of Mia (AR) pancreatic tumor cells. Overexpression of CEACAM6 in Capan2 pancreatic cancer cells enhances gemcitabine resistance, while siRNA-mediated inhibition of CEACAM6 expression in BxPC3 cells sensitizes them to drug chemo by regulating AKT activity in a Src-dependent manner (Duxbury et al., Cancer Res. 2004 Jun 1; 64(11): 3987-93). These effects correspond to increased invasiveness of high CEACAM6-expressing cells that exhibit c-src activity and matrix metalloproteinase (MMP9) expression (Duxbury et al., Br J Cancer. 2004 Oct 4; 91(7): 1384-90).

T-cell responses against tumor-associated antigens have been described in many tumors (Beckhove et al., J Clin Invest. 2004 Jul;114(1):67-76; Choi et al., Blood. 2005 Mar 1;105(5):2132-4; Sommerfeldt et al., Cancer Res. 2006 Aug 15;66(16):8258-65; Schmitz-Winnenthal et al., Cancer Res. 2005 Nov 1;65(21):10079-87.; Jäger et al., Proc Natl Acad Sci USA. 2000 Apr 25;97(9):4760-5; Romero et al., Adv Immunol. 2006;92:187-224) and often result in the accumulation of tumor-specific memory T cells in lymphoid organs or blood (Choi et al., Blood. 2005 Mar1;105(5):2132-4; Feuerer et al., Nat Med. 2001 Apr;7(4):452-8; Letsch et al., Cancer Res. 2003 Sep 1;63(17):5582-6). However, the ability of T cells to respond to autologous tumor cells is generally low (Horna and Sotomayor, Curr Cancer Drug Targets. 2007 Feb;7(1):41-53); Yang and Carbone, Adv Cancer Res. 2004;92:13-27). Many tumors have the ability to block T cell effector function, which limits the activity of tumor immunotherapy. For a wide range of cancers, T- cells have been shown to be unresponsive to tumor cells (Pardoll, Nat Immunol. 2012 Dec;13(12):1129-32).

CEACAM6 also contributes to the regulation of CD8+ T cell responses. Recently, Witzens-Harig et al. showed that treatment with anti-CEACAM6 mAbs or siRNA silencing CEACAM6 restored T cell reactivity against malignant plasma cells in multiple myeloma cells expressing several CEACAM family members, indicating a role for CEACAM6 in the regulation of CD8+ T cell responses (Witzens-Harig et al., Blood 2013 May 30; 121(22): 4493-503). To date, receptors for CEACAM6 on T cells have been identified. However, co-culture of CEACAM6-positive myeloma cells with T cells results in modulation of T cell signaling events, including activation of SHP phosphatase through CEACAM6 ligation (Lin and Weiss, J Cell Sci. 2001 Jan;114(Pt 2):243-4; Latour et al., Mol Cell Biol. 1997 Aug;17(8):4434-41; Wen et al., J Immunol. 2010 Dec 1;185(11):6413-9). CEACAM6 does not have intrinsic signaling capacity, and its inhibitory capacity is presumably mediated by binding to a receptor on the surface of T cells. This receptor may be, for example, CEACAM1, whose mechanisms for regulating innate and adaptive immune responses have been described. CEACAM1 (CD66a) has a cytoplasmic tail containing an immunoreceptor tyrosine-based inhibitory (ITIM) motif. CEACAM1 is stored in intracellular vesicles and is rapidly externalized and expressed on the T cell surface after T cell activation (24h to 72h), where it mediates the blockade of T cell effector function after binding to homophilic and heterophilic ligands expressed on target cells (Gray-Owen and Blumberg, Nat Rev Immunol. 2006 Jun;6(6):433-46). The nature of this binding is unknown and may be homophilic and heterophilic binding to other CEACAMs or binding to other components of the extracellular matrix, growth factor receptors, integrins, or cadherins. Homophilic interactions between CEACAM1 and CEACAM1 have been reported (Ortenberg et al., Mol Cancer Ther. 2012 Jun;11(6):1300-10). For example, heterophilic CEACAM interactions have been described between CEACAM1 and CEACAM5, and CEACAM6 and CEACAM8 (Cavallaro and Christofori, Nat Rev Cancer. 2004 Feb;4(2):118-32).

As described above, CEACAM6 is a very attractive target for therapeutic intervention in cancer immunotherapy. As shown, CEACAM6 is a member of a family of highly homologous proteins. Antibodies suitable for human therapy that alleviate the immunosuppression of CEACAM6 must therefore be able to distinguish CEACAM6 from other paralogous proteins, such as CEACAM1, CEACAM3, and CEACAM5, each of which exhibits distinct functions and tissue distributions, in order to restrict their mode of action to CEACAM6 and avoid undesirable side effects.

Since CEACAM6 is expressed not only on tumor cells but also on normal tissues (particularly granulocytes and epithelial cells such as lung and gastrointestinal cells - Chan and Stanners, Mol Ther. 2004 Jun;9(6):775-85; Strickland et al., J Pathol. 2009 Jul;218(3):380-90), it is very important to be able to predict the adverse side effect profile of therapeutic antibodies. This is all the more important because the expected mode of action will be inhibition of immunosuppression, i.e., immune activation, which can lead to serious harm (an event in the trial of the CD28 superagonist TGN1412; Suntharalingam et al., N Engl J Med. 2006 Sep 7;355(10):1018-28). Therefore, indirect effects on the immune system in addition to direct effects on granulocytes need to be carefully evaluated. To enable the development of human therapeutic antibodies and predictive preclinical tolerability testing, it is mandatory that the antibodies show relevant cross-reactivity with toxicologically relevant species, in the case of CEACAM6 in non-human primates, preferably Macaca fascicularis (cynomolgus).

As a prerequisite, therapeutic antibodies need to bind to human CEACAM6 on cells with high affinity, bind selectively to CEACAM6 (not to any paralogs), cross-react with monkey CEACAM6 with a monovalent _KD within an order of magnitude (to safely reflect binding on normal tissues in toxicology monkey models, even at low surface density under non-avidity-based binding conditions), bind to an epitope similar to that on human CEACAM6, be able to alleviate CEACAM6-mediated immunosuppression, be immunogenic in human therapy (i.e., human or humanized antibodies), and be sufficiently stable to allow clinical development, formulation, and storage as drugs over extended periods of time. The latter is important because it has been noted previously that physical degradation (particularly aggregation) can enhance the immune response to therapeutic proteins (Hermeling et al., Pharm Res. 2004 Jun;21(6):897-903), and aggregation is closely linked to the unfolding of IgG and its thermal stability (Vermeer and Norde, Biophys J. 2000 Jan;78(1):394-404).

Several anti-CEACAM6 antibodies exist. Most are non-human reagent antibodies, many of which are polyclonal. Their specificity and selectivity for human CEACAM6 and their cross-reactivity with monkey CEACAM6 are not disclosed or known in most cases.

Therapeutic antibodies against CEACAM6 are also known in the art. Some are not selective for human CEACAM6 (e.g., MN-3 from Immunomedics, Neo201/h16C3 from Neogenix; both also bind to human CEACAM5). The single-domain antibody 2A3 and its fusion variants have not been characterized for selectivity and cross-reactivity against monkey CEACAM6 (WO2012040824 and Niu et al., J Control Release. 2012 Jul 10; 161(1): 18-24).

No selective anti-CEACAM6 antibodies that significantly cross-react with monkey CEACAM6 have been disclosed (Strickland et al., J Pathol. 2009 Jul;218(3):380-90).

The murine antibody 9A6 (Genovac/Aldevron) is the only antibody described as being able to modulate the immunosuppressive activity of CEACAM6 (Witzens-Harig et al., Blood 2013 May 30;121(22):4493-503). 9A6 inhibits the immunosuppressive activity of CEACAM6, resulting in enhanced cytokine secretion by T cells in vitro and anti-tumor efficacy in vivo (Khandelwal et al., Poster Abstract 61, Meeting Abstract from 22nd Annual International Cancer Immunotherapy Symposium October 6-8, 2014, New York City, USA). Although its selectivity appears to be appropriate, its cross-reactivity with monkey CEACAM6 has not been previously characterized. Furthermore, its murine nature precludes direct therapeutic application in humans.

As shown in the Examples, antibody 9A6 binds to recombinant human CEACAM6, but no binding is detected to recombinant macaque or cynomolgus monkey CEACAM6. For comparison, Neo201-hIgG1 was also tested. This antibody exhibits high-affinity binding to both human and monkey CEACAM6. However, Neo201 binds to human CEACAM5 and CEACAM6 and is therefore not specific for CEACAM6.

In summary, there is a great need for therapeutic monoclonal antibodies that contain the following characteristics:

i. The antibody is a high affinity binder to human CEACAM6.

ii. The antibody is selective for CEACAM6 and does not bind to any paralogs, in particular CEACAM1, CEACAM3 and CEACAM5.

iii. The antibody cross-reacts with monkey CEACAM6 with a monovalent _KD within one order of magnitude.

iv. The antibody is non-immunogenic in human therapy, i.e. it is a human or humanized antibody.

v. The antibody is capable of alleviating CEACAM6-mediated immunosuppression.

Such antibodies do not exist in the prior art. 9A6, which binds to the N-terminal domain 1 of human CEACAM6, is the only known anti-CEACAM6 antibody capable of alleviating CEACAM6-mediated immunosuppression but lacks cross-reactivity with monkey CEACAM6, except as a mouse antibody. Neo201 binds to a different domain of CEACAM6 besides the N-terminal domain 1. ADCC-based therapeutic efficacy of Neo201-hIgG1 has been reported (Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR: Du et al., Cancer Res April 15, 2011; 71(8 Supplement):4582).

The present inventors hypothesized that alleviation of CEACAM6-mediated immunosuppression is associated with binding to the N-terminal domain 1. However, the generation of antibodies that bind to the N-terminal domain 1 of CEACAM6 poses a challenging selectivity problem.

The sequence alignment in Figure 1 shows a very high degree of similarity between the protein sequences of human CEACAM6 and those of human CEACAM3, human CEACAM5, and human CEACAM1 throughout the extracellular region. The region of interest (Domain 1 of human CEACAM6) is particularly similar to other CEACAMs, as also reflected in Table 7. Human CEACAM6 paralogs (e.g., CEACAM1, CEACAM3, and CEACAM5) are much more similar to human CEACAM6 than the cynomolgus monkey ortholog. In fact, only two positions in the N-terminal region of the primary sequence are identical between human and cynomolgus monkey CEACAM6, but differ in amino acids from other human paralogs (marked with asterisks in Figure 1).

Unexpectedly, the inventors were able to find a method to generate antibodies that contain all the desired selectivity and functional characteristics.

SUMMARY OF THE INVENTION

The present invention relates to antibodies, antigen-binding antibody fragments, or variants thereof that exhibit high affinity for human and cynomolgus macaque CEACAM6 proteins and do not significantly cross-react with the closely related human CEACAM1, human CEACAM3, and human CEACAM5. This means that the antibodies, antigen-binding antibody fragments, or variants thereof are selective for CEACAM6. The provided antibodies bind to the N-terminal domain 1, which is highly conserved among these proteins.

The anti-CEACAM6 antibodies of the present invention are capable of altering the cytokine profile of tumor-specific T cells in vitro toward a more cytotoxic and/or activated phenotype characterized by increased secretion of IFN-γ, IL-2, and/or TNF-α. Thus, the antibodies of the present invention are capable of alleviating CEACAM6-mediated immunosuppression and inducing immune activation, which ultimately leads to anti-tumor efficacy in vivo.

The antibodies or antigen-binding antibody fragments or variants thereof of the present invention interfere with the interaction between CEACAM6 and CEACAM1, which may be a mechanism for regulating innate and adaptive immune responses.

The antibodies of the present invention are therefore suitable for the treatment of cancer and its metastases, in particular tumors expressing CEACAM6, such as colorectal cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), pancreatic cancer, gastric cancer, breast cancer and multiple myeloma.

The present invention describes antibodies that differ from existing anti-CEACAM6 antibodies in that they bind to human and cynomolgus monkey CEACAM6 with a monovalent _KD within an order of magnitude (to safely reflect binding on normal tissues in toxicology monkey models, even at low surface densities under non-avidity-based binding conditions) and do not significantly cross-react with the closely related paralogs CEACAM1, CEACAM3 and CEACAM5. Therefore, these antibodies are suitable for preclinical toxicology studies in cynomolgus monkeys to assess their safety profile. Since CEACAM6 is expressed not only on tumor cells but also on normal tissues (particularly granulocytes, but also epithelial cells such as lung and gastrointestinal cells - Chan and Stanners, Mol Ther. 2004 Jun;9(6):775-85; Strickland et al., J Pathol. 2009 Jul;218(3):380-90), it is absolutely crucial to be able to predict the adverse side effect profile of therapeutic antibodies. This is entirely more important since the intended mode of action would be to inhibit immunosuppression, i.e. immune activation, which can lead to serious harms (an event in the trials of the CD28 superagonist TGN1412), and therefore indirect effects on the immune system in addition to direct effects on granulocytes need to be carefully evaluated.

Highly preferred anti-CEACAM6 antibodies of the present invention are described in Table 1 and are characterized by their structural features.

In some embodiments, the anti-CEACAM6 antibodies of the present invention bind to an epitope of human CEACAM6, wherein the epitope comprises one or more amino acid residues selected from Gln60, Asn61, Arg62, Ile63, Val83, Ile84, Gly85, Thr90, Ser127, Asp128, and Leu129 of SEQ ID NO: 179.

In certain embodiments, an anti-CEACAM6 antibody of the invention binds to an epitope of human CEACAM6, wherein the epitope comprises amino acid residues Gln60, Asn61, Arg62, Ile63, Val83, Ile84, Gly85, Thr90, Ser127, Aspl28, and Leu129 of SEQ ID NO: 179.

In some embodiments, the anti-CEACAM6 antibodies of the invention interact with, e.g., bind to, an epitope of human CEACAM6, wherein the epitope comprises one, two, three, four, five, eight, ten, fifteen, or more amino acid residues selected from Pro59, Gln60, Asn61, Arg62, Ile63, Gly64, Val83, Ile84, Gly85, Thr86, Gln88, Thr90, Pro91, Ile125, Ser127, Asp128, and Leu129 of SEQ ID NO: 179.

In certain embodiments, the anti-CEACAM6 antibodies of the invention interact with, e.g., bind to, an epitope of human CEACAM6, wherein the epitope comprises amino acid residues Pro59, Gln60, Asn61, Arg62, Ile63, Gly64, Val83, Ile84, Gly85, Thr86, Gln88, Thr90, Pro91, Ile125, Ser127, Asp128, and Leu129 of SEQ ID NO: 179.

The anti-CEACAM6 antibodies of the present invention can be co-administered with known drugs, and in some cases, the antibodies themselves can be modified. For example, the antibodies can be conjugated with cytotoxic agents, immunotoxins, toxin clusters, or radioisotopes to potentially further increase their efficacy.

The present invention further provides such antibodies, which constitute a tool for diagnosing malignant or dysplastic conditions in which CEACAM6 expression is elevated compared to normal tissue. Anti-CEACAM6 antibodies are provided that are conjugated to a detectable label. Preferred labels are radiolabels, enzymes, chromophores, or fluorophores.

The present invention also relates to polynucleotides encoding the antibodies or antigen-binding fragments thereof of the present invention, cells expressing the antibodies or antigen-binding fragments thereof of the present invention, methods for producing the antibodies or antigen-binding fragments thereof of the present invention, methods for inhibiting the growth of dysplastic cells using the antibodies or antigen-binding fragments thereof of the present invention, and methods for treating and detecting cancer using the antibodies or antigen-binding fragments thereof of the present invention.

The present invention also relates to isolated nucleic acid sequences, each of which can encode an antibody or antigen-binding fragment thereof that is specific for an epitope of CEACAM6. The nucleic acids of the present invention are suitable for the recombinant production of the antibody or antigen-binding antibody fragment. Accordingly, the present invention also relates to vectors and host cells containing the nucleic acid sequences of the present invention.

The compositions of the present invention can be used for therapeutic or prophylactic applications. Thus, the present invention includes pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier or excipient. In a related aspect, the present invention provides methods for treating a disorder or condition associated with the undesirable presence of cells expressing CEACAM6. In a preferred embodiment, the disorder is cancer. This method comprises the step of administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an antibody of the present invention as described or contemplated herein.

Furthermore, the present invention relates to methods for generating antibodies of this type. The present invention also provides instructions for using an antibody library to isolate one or more members of such a library that specifically bind to CEACAM6. Furthermore, the present invention provides instructions for immunizing mice to produce hybridoma cell lines that secrete antibodies that specifically bind to CEACAM6 and cross-react with cynomolgus macaque (Macaca fascicularis) CEACAM6. The present invention also provides instructions for humanizing murine antibodies that specifically bind to CEACAM6.

Description of the drawings

Figure 1: Protein sequence alignment of the extracellular regions of human CEACAM6 paralogs and cynomolgus monkey (Macaca fascicularis) CEACAM6 orthologs. Numbers indicate amino acid positions after removal of the signal peptide sequence. Positions in the primary sequence of the N-terminal region that are identical in human and cynomolgus monkey CEACAM6 but differ in the amino acid at that position in other human paralogs are marked with asterisks. The N-terminal domain 1 is boxed.

Figure 2: Amino acid sequences of the variable domains VL and VH of TPP-2971. Sequences grafted into the human framework are highlighted in underlined bold letters. CDRs according to the Kabat definition are written in italics. Grey shaded letters indicate sequence differences between TPP-3187 and TPP-2971.

Figure 3: Amino acid sequences of the variable domains VL and VH of TPP-3310 and TPP-3714. The sequences of the murine CDRs derived from TPP-2971 are highlighted in bold underlined letters. The CDRs according to the Kabat definition are written in italic letters. Antibodies TPP-3310 and TPP-3714 differ in two amino acids within the VH framework, which are highlighted in non-bold underlined letters.

Figure 4: Amino acid sequences of the variable domains VL and VH of TPP-3820 and TPP-3821. The sequences of the murine CDRs derived from TPP-3187 are highlighted in bold underlined letters. The CDRs according to the Kabat definition are written in italic letters. Antibodies TPP-3820 and TPP-3821 differ in two amino acids within the VH framework, which are highlighted in non-bold underlined letters.

Figure 5: In vitro pharmacological effects of anti-CEACAM6 antibodies on IFN-γ secretion and the extent of IFN-γ secretion by survivin peptide-specific T cells. A+B. IFN-γ ELISpot assay of survivin peptide-specific T cells and KS tumor cells. 10,000 KS tumor cells were cocultured with 2,500 survivin T cells for 20 hours. The antibody concentration in the coculture was 30 µg/ml. C. IFN-γ ELISA assay of survivin peptide-specific T cells and KS tumor cells. 10,000 KS tumor cells were cocultured with 20,000 survivin T cells for 20 hours. The antibody concentration in the coculture was 30 µg/ml. The X-axis shows the different conditions tested: in A: 1 = 10,000 KS cells; 2 = 2,500 T cells; 3 = no antibody treatment; 4 = isotype-matched antibody control; 5 = TPP-3470 (9A6-hIgG2) 6 = TPP-3323; in B: 1 = 10,000 KS cells; 2 = 2,500 T cells; 3 = no antibody treatment; 4 = isotype-matched antibody control; 5 = TPP-3470 (9A6-hIgG2) 6 = TPP-3310; 7 = TPP-3707; in C: 1 = 10,000 KS cells; 2 = 20,000 T cells; 3 = no antibody treatment; 4 = isotype-matched antibody control; 5 = TPP-3470 (9A6-hIgG2) 6 = =TPP-3310; 7 =TPP-3707; Y-axis corresponds to IFN-γ spot counts per well (in A and B) or IFN-γ concentration in pg/ml (in C). Asterisks indicate statistically significant results based on an unpaired, two-tailed Student's t-test. Error bars represent SEM.

Figure 6: In vitro pharmacological effects of anti-CEACAM6 antibodies on cytokine secretion (IFN-γ, IL-2, and TNF-α) by survivin peptide-specific T cells. A. IFN-γ Luminex analysis. B. IL-2 Luminex analysis. C. TNFα Luminex analysis. Luminex cytokine analysis of survivin peptide-specific T cells and KS tumor cells. 10,000 KS tumor cells were cocultured with 20,000 survivin T cells for 20 hours. The antibody concentration in the coculture was 30 µg/ml. The X-axis depicts the different conditions tested: 1 = 10,000 KS cells; 2 = 20,000 T cells; 3 = no antibody treatment; 4 = isotype-matched antibody control; 5 = TPP-3470 (9A6-hIgG2); 6 = TPP-3310; 7 = TPP-3707; the Y-axis corresponds to cytokine concentration in pg/ml.

Figure 7: Effect of anti-CEACAM6 antibodies on in vivo tumor growth. 2 x 10 ⁶ KS breast cancer cells were inoculated subcutaneously. Tumor antigen-specific T cells (survivin-peptide specific) were injected intravenously on days 23 and 27. 200 μg of anti-CEACAM6 antibody or a matched isotype control was administered intraperitoneally on days 22, 24, 26, and 28. Tumor growth was assessed every 2-3 days. Error bars represent mean average (SEM). Y-axis = tumor surface (mm ² ); X-axis = day; TC = survivin-peptide-specific T cells. 1 = PBS-treated; 2 = T cells and isotype-matched antibody control-treated; 3 = T cells and TPP-3470 (9A6-hIgG2)-treated; 4 = T cells and TPP-3310-treated; 5 = T cells and TPP-3707-treated.

Figure 8: Annotated sequences of preferred anti-CEACAM6 antibodies of the invention. Protein and DNA sequences of the heavy and light chains of IgG, as well as the VH and VL regions of selected antibodies, are provided. Important regions (VH and VL regions, as well as CDR regions (H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, L-CDR3) in full-length IgG) are annotated below the sequences.

Figure 9: Cartoon representation of a single N-terminal domain 1 of human CEACAM6 (TPP-1794, white) bound to the Fab fragment APP-1574 (heavy and light chains coloured dark and light grey, respectively).

Figure 10: Details of the protein interface shown in Figure 9. Selected residues are depicted in stick representation and colored as in Figure 9. Numbering corresponds to TPP-1794 (SEQ-ID NO: 169)

Figure 11: xCELLigence cytotoxicity assay using survivin-peptide-specific CD8 ⁺ T cells.

A. 20,000 KS breast cancer tumor cells were cocultured with 20,000 survivin-specific T cells. B. 40,000 HCT-116-hC6 tumor cells were cocultured with 20,000 survivin-specific T cells. Cytotoxicity was monitored for ~100 hours. Antibodies were used at a final concentration of 30 µg/ml: #1, time point of T cell addition; #2, tumor cells alone; #3, no antibody; #4, isotype-matched antibody control; #5, anti-PD-L1 Ab as human IgG2; #6, TPP-34709A6 Ab as human IgG2; #7 TPP-3310 hIgG2. Asterisks indicate statistically significant results based on an unpaired, two-tailed Student's t-test. X, x-axis, time (hours); Y, y-axis, normalized cell index.

Figure 12: xCELLigence cytotoxicity assay using patient-derived T cells (TIL-12) from pancreatic cancer.

A and B: 10,000 HCC2935 tumor cells were co-cultured with 50,000 pancreatic cancer infiltrating lymphocytes (TIL-12). Cytotoxicity was monitored for ~150 hours. Anti-CD3 x EpCAM bispecific mAb (0.25 ng/ml) was added to the co-culture to direct T cells against tumor cells independently of HLA.

A: #1, T cells added; #2 tumor cells only; #3, no antibody; #4, isotype-matched antibody control; #5, anti-PD-L1 Ab as human IgG2; #6, TPP-3470; #7 TPP-3310; antibodies were used at 30 μg/ml.

B: Concentration dependence of TPP-3310-mediated effects: #1, T cell addition; #2, tumor cells only; #3, TPP-3310, 0.07 µg/ml; #4, TPP-3310, 0.02 µg/ml; #5, isotype-matched antibody control, 50 µg/ml; #6, TPP-3310, 0.021 µg/ml; #7, TPP-3310, 0.062 µg/ml; #8, TPP-3310, 1.85 µg/ml; #9, TPP-3310, 5.5 µg/ml; #10, TPP-3310, 16.67 µg/ml; #11, TPP-3310, 50 µg/ml;

X, x-axis, time (hours); Y, Y-axis, normalized cell index.

Detailed Description of the Invention

The present invention is based on the discovery of novel antibodies that have specific affinity for CEACAM6 and can deliver therapeutic benefit to a subject.The antibodies of the invention (which may be human, humanized or chimeric) can be used in a number of situations as will be more fully described herein.

definition

Unless otherwise defined, all technical and scientific terms used herein have the meanings generally understood by those of ordinary skill in the art to which the present invention belongs. However, the following references can provide general definitions of many terms used in the present invention for those of ordinary skill in the art to which the present invention relates, and can be referenced and used, as long as such definitions are consistent with the meanings generally understood in the art. Such references include, but are not limited to: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition 1994); The Cambridge Dictionary of Science and Technology (Walker edited, 1988); Hale & Marham, The Harper Collins Dictionary of Biology (1991); and Lackie et al., The Dictionary of Cell & Molecular Biology (3rd edition 1999); and Cellular and Molecular Immunology, edited by Abbas, Lichtman and Pober, 2nd edition, W.B. Saunders Company. Any additional technical resources that can be used to provide definitions of the terms used herein with the meanings generally understood in the art that can be queried by those of ordinary skill in the art. For the purposes of the present invention, the following terms are further defined. Additional terms are defined elsewhere in this specification. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a gene" refers to one or more genes and includes equivalents thereof known to those skilled in the art, and so forth.

The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

Amino acids may be referred to herein by either their well-known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides may be referred to by their commonly accepted single-letter codes.

As used herein, "CEACAM6" means "carcinoembryonic antigen-related cell adhesion molecule 6," also known as "CD66c" (cluster of differentiation 66c) or nonspecific cross-reactive antigen or NCA or NCA-50/90. CEACAM6 is a glycosylphosphatidylinositol (GPI)-linked cell surface protein involved in cell-cell adhesion. CEACAM6 is highly expressed on the surface of various tumor cells, such as colon cancer, pancreatic cancer, breast cancer, and lung cancer.

The reference sequence of human CEACAM6 can be obtained from the UniProtKB/Swiss-Prot database as accession number P40199.3 (SEQ-ID NO:179 = TPP-4639), including the signal peptide (positions 1-34) and the propeptide chain (positions 321-344). A single nucleotide polymorphism (G to V exchange) has been observed at position 239. The mature extracellular domain of human CEACAM6 consists of amino acids at positions 35-320 of SEQ-ID No: 179.

Human CEACAM6 (SEQ-ID NO: 179)

.

The cynomolgus monkey (Macaca fascicularis) protein sequence of CEACAM6 was deduced by the inventors and is represented by TPP-4189 (SEQ-ID No: 177). The mature extracellular domain of cynomolgus monkey CEACAM6 consists of amino acids at positions 35-320 of SEQ-ID No: 177.

Cynomolgus macaque (Macaca fascicularis) CEACAM6 (SEQ-ID NO: 177)

.

The domain organization of human and cynomolgus macaque (Macaca fascicularis) CEACAM6 is as follows (based on UniProtKB/Swiss-Prot database sequences, accession numbers P40199.3 and SEQ-ID NO: 179 = TPP-4639 & SEQ-ID No: 177 = TPP-4189, respectively):

The full-length human CEACAM1 protein is available from the UniProtKB/Swiss-Prot database under accession number P13688.2 (SEQ-ID No: 173 = TPP-4185). The mature extracellular domain of human CEACAM1 consists of amino acids at positions 35-428 of SEQ-ID No: 173.

Human CEACAM1 (SEQ-ID NO:173)

.

The full-length human CEACAM3 protein is available from the UniProtKB/Swiss-Prot database under accession number P40198.2 (SEQ-ID No: 175 = TPP-4187). The mature extracellular domain of human CEACAM3 consists of amino acids at positions 35-155 of SEQ-ID No: 175.

Human CEACAM3 (SEQ-ID NO: 175)

.

The full-length human CEACAM5 protein is available from the UniProtKB/Swiss-Prot database under accession number P06731.3 (SEQ-ID No: 176 = TPP-4188). The mature extracellular domain of human CEACAM5 consists of amino acids at positions 35-685 of SEQ-ID No: 176.

Human CEACAM5 (SEQ-ID NO: 176)

.

The terms "anti-CEACAM6 antibody" and "antibody that binds to CEACAM6" refer to antibodies that bind to CEACAM6 with sufficient affinity to allow the antibody to be used as a diagnostic and/or therapeutic agent targeting CEACAM6. In one embodiment, the extent of binding of the anti-CEACAM6 antibody to unrelated, non-CEACAM6 proteins is less than about 5%, preferably less than about 2%, of the binding of the antibody to CEACAM6, as measured, for example, by surface plasmon resonance (SPR). In certain embodiments, the antibody that binds to CEACAM6 has a dissociation constant (KD) of ≤ 1 µM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., 10 ⁻⁸ M or less, e.g., 10 ⁻⁸ M to 10 ⁻¹³ M, e.g., 10 ⁻⁹ M to 10 ⁻¹³ _M ). In certain embodiments, an anti-CEACAM6 antibody binds to an epitope of CEACAM6 that is conserved among CEACAM6 from different species.

As used herein, the term "antibody" refers to an immunoglobulin molecule, which is preferably composed of four polypeptide chains: two heavy (H) chains and two light (L) chains (which are usually interconnected by disulfide bonds). Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region may contain, for example, three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain (CL). The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL is usually composed of three CDRs and up to four FRs, which are arranged, for example, in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

As used herein, the term "complementarity determining region (CDR; e.g., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain whose presence is required for antigen binding. Each variable domain typically has three CDR regions, referred to as CDR1, CDR2, and CDR3. Each CDR can comprise amino acid residues from a "complementarity determining region" as defined by Kabat (e.g., approximately residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and approximately residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; (Kabat et al., Sequences of Proteins of Immulological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or those residues from a "hypervariable loop" (e.g., approximately residues 26-32 in the light chain variable domain). In some cases, the CDRs may comprise amino acids from both the CDR regions and the hypervariable loops as defined by Kabat.

Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be divided into different "classes". There are five major classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The preferred immunoglobulin class for use in the present invention is IgG.

The heavy chain constant domains corresponding to the different antibody classes are referred to as [α], [δ], [ε], [γ], and [μ], respectively. The subunit structures and three-dimensional configurations of the different immunoglobulin classes are well known. Antibodies as used herein are conventionally referred to as antibodies and their functional fragments.

"Functional fragments" or "antigen-binding antibody fragments" of antibodies/immunoglobulins are defined herein as fragments of antibodies/immunoglobulins that retain an antigen-binding region (e.g., the variable region of IgG). The "antigen-binding region" of an antibody is typically found in one or more hypervariable regions of an antibody, such as the CDR1, CDR2, and/or CDR3 regions; however, the variable "framework" regions may also play an important role in antigen binding, such as by providing a scaffold for the CDRs. Preferably, the "antigen-binding region" comprises at least amino acid residues 4-103 of the variable light (VL) chain and at least amino acid residues 5-109 of the variable heavy (VH) chain, more preferably amino acid residues 3-107 of VL and amino acid residues 4-111 of VH, and particularly preferred are complete VL and VH chains (amino acid positions 1-109 of VL and amino acid positions 1-113 of VH; numbering according to WO 97/08320).

"Functional fragments,""antigen-binding antibody fragments," or "antibody fragments" of the present invention include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') ₂ , and Fv fragments; diabodies; single-domain antibodies (DAb), linear antibodies; single-chain antibody molecules (scFv), and multispecific (such as bispecific and trispecific) antibodies formed from antibody fragments (CA K Borrebaeck, ed. (1995) Antibody Engineering (Breakthroughs in Molecular Biology), Oxford University Press; R. Kontermann & S. Duebel, eds. (2001) Antibody Engineering (Springer Laboratory Manual), Springer-Verlag). Antibodies other than "multispecific" or "multifunctional" antibodies are understood to have identical binding sites. F(ab') ₂ or Fab can be engineered to minimize or completely eliminate the intermolecular disulfide interactions that exist between the _CH1 and _CL domains.

The term "Fc region" herein is used to define the C-terminal region of the immunoglobulin heavy chain containing at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not exist. Unless otherwise specified in this article, the numbering of the amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

Variants of antibodies or antigen-binding antibody fragments contemplated in the present invention are molecules in which the binding activity of the antibody or antigen-binding antibody fragment is maintained.

"Binding proteins" contemplated in the present invention are, for example, antibody mimetics, such as Affibodies, Adnectins, Anticalins, DARPins, Avimers, Nanobodies (reviewed in Gebauer M. et al., Curr. Opinion in Chem. Biol. 2009; 13:245-255; Nuttall S.D. et al., Curr. Opinion in Pharmacology 2008; 8:608-617).

"Human" antibodies or their antigen-binding fragments are defined herein as antibodies or their antigen-binding fragments that are not chimeric (e.g., non-"humanized") and are not from (completely or partially) non-human species. Human antibodies or their antigen-binding fragments may be derived from humans or may be synthetic human antibodies. "Synthetic human antibodies" are defined herein as antibodies having sequences derived entirely or partially on a computer chip from synthetic sequences based on analysis of known human antibody sequences. The design of human antibody sequences or their fragments on a computer chip can be achieved, for example, by analyzing a database of human antibody or antibody fragment sequences and utilizing the data obtained therefrom to design polypeptide sequences. Another example of a human antibody or its antigen-binding fragment is a human antibody or its antigen-binding fragment encoded by a nucleic acid separated from a library of antibody sequences from human sources (e.g., this library is based on antibodies taken from human natural sources). Examples of human antibodies include antibodies described in, for example, Söderlind et al., Nature Biotech. 2000, 18:853-856.

A "humanized antibody" or a humanized antigen-binding fragment thereof is defined herein as a humanized antibody or a humanized antigen-binding fragment thereof that: (i) is derived from a non-human source (e.g., a transgenic mouse carrying a xenogeneic immune system), wherein the antibody is based on a human germline sequence; (ii) wherein the amino acids of the framework regions of the non-human antibody are partially converted to human amino acid sequences by genetic engineering, or (iii) is CDR-grafted, wherein the CDRs of the variable domain are from a non-human source, while one or more frameworks of the variable domain are of human origin, and the constant domains (if any) are of human origin.

A "chimeric antibody" or antigen-binding fragment thereof is defined herein as one in which the variable domains are derived from a non-human source and some or all of the constant domains are derived from a human source.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a basic homogeneous antibody population, that is, except for the possible mutation (such as naturally occurring mutation) that can exist in a small amount, the individual antibodies constituting the population are identical. Therefore, the term "monoclonal" shows that antibody characteristics are not a mixture of discrete antibodies. Contrary to the polyclonal antibody preparations that generally include different antibodies for different determinants (epitopes), each monoclonal antibody of the monoclonal antibody preparation is directed to a single determinant on the antigen. Except for its specificity, the monoclonal antibody preparation is advantageous because they are not contaminated by other immunoglobulins conventionally. The term "monoclonal" should not be construed as needing to produce antibody by any ad hoc method. The term monoclonal antibody particularly includes chimeric antibodies, humanized antibodies and human antibodies.

An "isolated" antibody is one that has been identified and separated from components of the cells in which it is expressed. Contaminant cellular components are substances that may interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.

An "isolated" nucleic acid is one that has been identified and separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

As used herein, an antibody that "specifically binds" to an antigen of interest (e.g., a tumor-associated polypeptide antigen target), is "specific for" or "specifically recognizes" an antigen of interest is an antibody that binds to the antigen with sufficient affinity so that the antibody can be used as a therapeutic agent in cells or tissues targeted to express the antigen and does not significantly cross-react with other proteins or does not significantly cross-react with proteins other than orthologs and variants (e.g., mutant forms, splice variants, or proteolytic truncated forms) of the aforementioned antigen target. For example, an antibody or antigen-binding fragment thereof having a monovalent _KD for an antigen of less than about 10-4 M, or less than about ^10-5 M, or less than about 10-6 M, or less than about 10-7 M, or less than about 10-8 M, or less than about 10-9 M, or less than about ^10-10 M, or less than about ^10-11 M, or less than about ^10-12 M or less can be shown as "specifically recognizing" or "specifically binding" a particular polypeptide or an epitope on a ^particular polypeptide target, or being "specific for ^" a particular polypeptide or an epitope on a particular polypeptide target, as used herein. An antibody ^" specifically binds" to, is ^" specific for," or "specifically recognizes" an antigen if the antibody is able to distinguish the antigen from one or more reference antigens. In its most general ^form , "specific binding,""specificallybinds,""specificfor," or "specifically recognizes" refers to the ability of an antibody to distinguish between an antigen of interest and an unrelated antigen, as determined, for example, by one of the following methods. Such methods include, but are not limited to, surface plasmon resonance (SPR), Western blot, ELISA test, RIA test, ECL test, IRMA test and peptide scanning. For example, standard ELISA assay can be implemented. Scoring can be implemented by standard color development (e.g., secondary antibody with horseradish peroxidase and tetramethylbenzidine with hydrogen peroxide). The reaction in certain wells is scored by, for example, an optical density at 450 nm. A typical background (= negative reaction) can be 0.1 OD; a typical positive reaction can be 1 OD. This means that the difference between positive/negative is greater than 5 times, 10 times, 50 times and preferably greater than 100 times. Typically, a single reference antigen is not used, but a collection of about three to five unrelated antigens, such as milk powder, BSA, transferrin, etc., is used to determine binding specificity.

"Binding affinity" or "affinity" refers to the strength of the sum of the non-covalent interactions between a single binding site of a molecule and its binding partner. Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The dissociation constant " _KD " is generally used to describe the affinity between a molecule (such as an antibody) and its binding partner (such as an antigen), i.e., how tightly a ligand binds to a particular protein. Ligand-protein affinity is influenced by non-covalent intermolecular interactions between the two molecules. Affinity can be measured by common methods known in the art, including those described herein. In one embodiment, the "KD" or "KD value" according to the present invention is measured by using surface plasmon resonance assays using a suitable device, including but not limited to a Biacore instrument such as Biacore _T100 , Biacore T200, Biacore 2000, Biacore 4000, Biacore 3000 (GE Healthcare Biacore, Inc.) or a ProteOn XPR36 instrument (Bio- _Rad Laboratories, Inc.).

As used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitope determinants are usually composed of chemically active surface groupings of molecules (such as amino acids or sugar side chains or combinations thereof) and usually have specific three-dimensional structural characteristics as well as specific charge characteristics.

An "antibody that binds to the same epitope as a reference antibody" or an antibody that "competes for binding" with a reference antibody means that it blocks binding of the reference antibody to its antigen by 10%, 20%, 30%, 40%, 50% or more in a competition assay, whereas, conversely, the reference antibody blocks binding of the antibody to its antigen by 10%, 20%, 30%, 40%, 50% or more in a competition assay. Exemplary competition assays are provided herein.

An "antibody that binds to an epitope of a target protein Z, wherein the epitope comprises amino acid residues X1, X2, X3, ..." is an antibody that, after binding of the antibody to its target protein, comprises atoms within 5 Å, preferably within 4 Å, of the atoms of amino acid residues X1, X2, X3, ... of the target protein Z. Such epitopes can be determined using X-ray crystal structures as exemplified in Example 16.

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fcγ receptors (FcγRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) causes these cytotoxic effector cells to specifically bind to target cells carrying the antigen, and then, for example, kill the target cells with cytotoxins. In order to evaluate the ADCC activity of the target antibody, an in vitro ADCC assay, such as that described in U.S. Patent No. 5,500,362 or 5,821,337 or U.S. Patent No. 6,737,056 (Presta), can be performed. Useful effector cells for such assays include PBMCs and NK cells.

"Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by binding of the first component of the complement system (Clq) to an antibody (of the appropriate subclass) that binds to its cognate antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996). Polypeptide variants with altered Fc region amino acid sequences (polypeptides having variant Fc regions) and increased or decreased Clq binding are described, for example, in U.S. Patent No. 6,194,551 B1 and WO 1999/51642.

As used herein, "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radiolabel. The naked antibody can be present in a pharmaceutical composition.

The term "immunoconjugate" (interchangeably referred to as "antibody-drug conjugate" or "ADC") refers to an antibody conjugated to one or more cytotoxic agents or cytostatic agents, such as chemotherapeutic agents, drugs, growth inhibitors, toxins (e.g., protein toxins of bacterial, fungal, plant or animal origin, enzymatically active toxins or fragments thereof) or radioactive isotopes (i.e., radioconjugates). Immunoconjugates have been used in cancer therapy for local delivery of cytotoxic agents, i.e., drugs that kill or inhibit the growth or proliferation of cells (e.g., Liu et al., Proc Natl. Acad. Sci. (1996), 93, 8618-8623). Immunoconjugates allow targeted delivery of the drug moiety to the tumor and accumulation within the cell therein, where systemic administration of the unconjugated drug can result in unacceptable toxicity levels to normal cells and/or tissues. Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin. The toxins may exert their cytotoxic effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.

" percent (%) sequence identity " about reference polynucleotide or peptide sequence is defined as respectively after comparing sequences and introducing gap (if necessary) to reach maximum percent sequence identity, the percentage of nucleic acid or amino acid residue in the candidate sequence respectively identical with the nucleic acid or amino acid residue in the reference polynucleotide or peptide sequence.Conservative substitutions are not considered as the part of sequence identity.Preferably, gapless comparison.Comparison for determining percent amino acid sequence identity purpose can be realized in the various ways within the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine the appropriate parameters for comparing sequences, including any algorithm required for maximum comparison that is reached within the total length of the sequence being compared.

"Sequence homology" indicates the percentage of identical amino acids or amino acids representing conservative amino acid substitutions.

The terms "mature antibody" or "mature antigen-binding fragment," such as mature Fab variants, include derivatives of antibodies or antibody fragments that exhibit stronger binding (i.e., binding with increased affinity) to a given antigen, such as the extracellular domain of a target protein. Maturation is the process of identifying a small number of mutations within, for example, the six CDRs of an antibody or antibody fragment that result in this increased affinity. The maturation process is a combination of molecular biological methods for introducing mutations into antibodies and screening for identifying improved binding agents.

An "antagonistic" antibody or "blocking" antibody is an antibody that significantly inhibits (partially or completely) the biological activity of the antigen to which it binds.

An "agonistic" antibody or an antibody with "agonistic activity" is an antibody that binds (partially or fully) to its target and induces activation of the respective target, e.g., causes (partially or fully) activation of a signaling pathway or biological effect mediated by the respective target. As used herein, an "agonistic" antibody or an antibody with "agonistic activity" is an antibody that can mimic at least one functional activity of a polypeptide of interest.

The term "pharmaceutical formulation"/"pharmaceutical composition" refers to a preparation that is in such form as to permit the biological activity of the active ingredient contained therein to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered.

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants," "transformed cells," "transfectants," "transfected cells," and "transduced cells," which include the initially transformed/transfected/transduced cell and progeny derived therefrom, without regard to the number of generations. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny that have been screened or selected for the same function or biological activity as in the initially transformed cell are included herein.

Antibodies of the present invention

The present invention relates to antibodies or antigen-binding antibody fragments or variants thereof that specifically bind to human CEACAM6 and cynomolgus monkey CEACAM6 and thus do not significantly cross-react with the closely related human CEACAM1, human CEACAM3 and human CEACAM5.

The antibodies or antigen-binding antibody fragments or variants thereof of the present invention specifically bind to the mature extracellular domain of CEACAM6 and the mature extracellular domain of cynomolgus monkey CEACAM6, and do not significantly cross-react with the mature extracellular domains of the closely related human CEACAM1, human CEACAM3, and human CEACAM5. The mature extracellular domain may be a portion of the full-length protein expressed on the cell surface as well as a soluble protein (naturally occurring or recombinantly expressed).

The antibodies of the present invention, or their antigen-binding antibody fragments or variants thereof, specifically bind to proteins comprising the mature extracellular domain of human CEACAM6 and/or the mature extracellular domain of cynomolgus monkey CEACAM6, and do not significantly cross-react with proteins comprising only the mature extracellular domain of the closely related human CEACAM1 and/or human CEACAM3 and/or human CEACAM5.

One embodiment of the present invention is to provide antibodies or antigen-binding antibody fragments or variants thereof that are specific for human and cynomolgus monkey CEACAM6, which means that the antibodies cross-react with human and cynomolgus monkey CEACAM6.

One embodiment of the present invention is to provide antibodies or antigen-binding antibody fragments or variants thereof that are selective for CEACAM6, meaning that they do not significantly cross-react with the closely related CEACAM1, CEACAM3 and CEACAM5.

Another embodiment of the present invention is to provide an antibody, or antigen-binding antibody fragment thereof, or variant thereof, that binds to human CEACAM6 and cross-reacts with CEACAM6 of another species (including but not limited to monkey) with similar affinity. Preferably, the other species is a non-human primate, such as, for example, cynomolgus monkeys, macaques, orangutans, gorillas, and chimpanzees. Most preferably, the antibody, or antigen-binding antibody fragment thereof, or variant thereof binds to human CEACAM6 and cross-reacts with cynomolgus monkey CEACAM6.

A monoclonal antibody that binds to antigen 1 (Ag1) "cross-reacts" with antigen 2 (Ag2) when the _EC50 and/or _KD values for the two antigens are in a similar range. In the present disclosure, a monoclonal antibody that binds to Ag1 cross-reacts with Ag2 when the ratio of the affinity for Ag1 to the affinity for Ag2 is equal to or less than 10 (≤ 10) and equal to or greater than 0.1 (≥ 0.1), meaning that the affinities for Ag1 and Ag2 do not differ by more than 10-fold (monovalent _KDs within one order of magnitude), provided that the affinities are measured using the same method for both antigens in the same experimental setup.

Thus, the ratio of the affinity of the antibodies according to the present invention for human CEACAM6 to the affinity for cynomolgus monkey CEACAM6 is equal to or less than 10 (≤ 10) and equal to or greater than 0.1 (≥ 0.1), which means that the affinities for human and cynomolgus monkey CEACAM6 do not differ by more than 10-fold (affinity is within one order of magnitude of monovalent _KD ). Therefore, the antibodies of the present invention, or antigen-binding antibody fragments thereof, or variants thereof, according to the present invention can be used in toxicology studies in monkeys, as the toxicity profiles observed in monkeys will be relevant to the potential adverse effects expected in humans.

A monoclonal antibody that binds to antigen 1 (Ag1) "does not significantly cross-react" with antigen 3 (Ag3) when the affinities for the two antigens are very different. If the binding response (binding signal measured in the assay) is too low, the affinity for Ag3 may not be measurable. In the present application, a monoclonal antibody that binds to Ag1 "does not significantly cross-react" with Ag3 when the binding response of the monoclonal antibody to Ag3 (binding signal measured in the assay) is less than 5%, preferably less than 2%, of the binding response of the same monoclonal antibody to Ag1 under the same experimental setup and the same antibody concentration. In practice, the antibody concentration used can be the EC ₅₀ or K _D or the concentration required to reach a saturation plateau obtained with Ag1.

According to the present invention, the antibody or antigen-binding antibody fragment or variant thereof does not significantly cross-react with human CEACAM1, human CEACAM3 and human CEACAM5.

A preferred embodiment of the present invention has an affinity for human CEACAM6 and cynomolgus monkey CEACAM6 of ≤ 400 nM, preferably ≤ 200 nM, or preferably ≤ 100 nM as determined as monovalent affinity to recombinant CEACAM6 (see Example 2), as shown in Tables 13, 18 and 20.

According to the present invention, the antibody or antigen-binding antibody fragment or variant thereof binds to human CEACAM6 domain 1 (represented by amino acids 35-142 of SEQ-ID No: 179) and cynomolgus monkey CEACAM6 domain 1 (represented by amino acids 35-142 of SEQ-ID No: 177) with an affinity within one order of magnitude of the monovalent _KD . CEACAM6 domain 1 is also known as the N domain.

One embodiment of the present invention is to provide an antibody, or antigen-binding antibody fragment, or variant thereof, that specifically binds to human CEACAM6 domain 1 (represented by amino acids 35-142 of SEQ-ID No: 179) and specifically binds to cynomolgus monkey CEACAM6 domain 1 (represented by amino acids 35-142 of SEQ-ID No: 177) with an affinity (monovalent K _D ) within an order of magnitude. Highly preferred are antibodies, or antigen-binding antibody fragments, or variants thereof, that have an affinity of K _D ≤ 100 nM for both human CEACAM6 domain 1 and cynomolgus monkey CEACAM6 domain 1.

The antibodies or antigen-binding antibody fragments or variants thereof of the present invention specifically bind to a protein comprising human CEACAM6 domain 1 (represented by amino acids 35-142 of SEQ-ID NO: 179) and/or cynomolgus monkey CEACAM6 domain 1 (represented by amino acids 35-142 of SEQ-ID NO: 177).

The antibodies, or antigen-binding antibody fragments, or variants thereof, of the present invention specifically bind to a protein comprising human CEACAM6 domain 1 (represented by amino acids 35-142 of SEQ-ID NO: 179) and/or cynomolgus monkey CEACAM6 domain 1 (represented by amino acids 35-142 of SEQ-ID NO: 177), and do not significantly cross-react with the closely related human CEACAM1, human CEACAM3, and human CEACAM5.

The antibodies or antigen-binding antibody fragments or variants thereof of the present invention specifically bind to proteins comprising human CEACAM6 domain 1 (represented by amino acids 35-142 of SEQ-ID NO: 179) and/or cynomolgus monkey CEACAM6 domain 1 (represented by amino acids 35-142 of SEQ-ID NO: 177), and do not significantly cross-react with proteins comprising only the mature extracellular domains of the closely related human CEACAM1 and/or human CEACAM3 and/or human CEACAM5.

One embodiment of the present invention is to provide an antibody or antigen-binding antibody fragment or variant thereof that specifically binds to the mature extracellular domain of CEACAM6 and the mature extracellular domain of cynomolgus monkey CEACAM6 and competes with the 9A6 antibody (Genovac/Aldevron) for binding to human CEACAM6 and does not significantly cross-react with the mature extracellular domains of the closely related human CEACAM1, human CEACAM3 and human CEACAM5.

One embodiment of the present invention is to provide an antibody, or an antigen-binding antibody fragment thereof, or a variant thereof, that specifically binds to human CEACAM6 domain 1 (represented by amino acids 35-142 of SEQ-ID NO: 179) and cynomolgus monkey CEACAM6 domain 1 (represented by amino acids 35-142 of SEQ-ID NO: 177), and competes with the 9A6 antibody (Genovac/Aldevron) for binding to human CEACAM6.

One embodiment of the present invention provides antibodies, or antigen-binding antibody fragments or variants thereof, that specifically bind to human CEACAM6 and cynomolgus monkey CEACAM6 and interfere with the interaction between CEACAM6 and CEACAM1. An antibody interferes with the interaction between CEACAM6 and CEACAM1 when, in a typical binding assay with CEACAM1 provided in the Examples, the binding signal of a preformed antibody-CEACAM6 complex is reduced by greater than 20%, preferably greater than 50%, compared to the binding signal of the CEACAM6 protein alone. Interference with the interaction between CEACAM6 and CEACAM1 may be a mechanism for regulating both innate and adaptive immune responses.

One embodiment of the present invention is to provide antibodies, or antigen-binding antibody fragments thereof, or variants thereof, that specifically bind to human CEACAM6 and cynomolgus monkey CEACAM6 and have immunomodulatory activity. "Immunomodulation" refers to the adjustment of an immune response to a desired level, such as induction of immunoprotection, immunosuppression, or immune tolerance. "Immunomodulatory antibody" drugs are immune response modulators that stimulate or inhibit an immune response for the treatment of a disease, such as cancer, or for anti-inflammatory purposes. The present invention provides anti-CEACAM6 immunomodulatory antibodies, as shown in Example 11, that block CEACAM6, an immune cell inhibitory ligand on cancer cells and potentially other cells (including components of the immune system), resulting in changes in immune marker expression profiles and T cell activation status toward anti-tumor T cell reactivation and an effective anti-cancer immune response. Antibodies that bind to domain 1 of CEACAM6 are preferred.

One embodiment of the present invention is to provide antibodies, or antigen-binding antibody fragments thereof, or variants thereof, that specifically bind to human CEACAM6 and cynomolgus monkey CEACAM6 and are capable of alleviating CEACAM6-mediated immunosuppression of tumor antigen-specific T cells, as measured by IFN-γ secretion by tumor antigen-specific T cells or the number of IFN-γ-secreting activated T cells. An anti-CEACAM6 antibody is capable of alleviating CEACAM6-mediated immunosuppression of tumor antigen-specific T cells if, in a co-culture of tumor-specific T cells with tumor cells, the antibody produces an increase in IFN-γ secretion by >1.2-fold, preferably >1.5-fold, compared to a control sample. Preferably, the tumor antigen-specific T cells are CD8 ⁺ T cells. Preferred antibodies are those that do not significantly cross-react with the closely related CEACAM1, CEACAM3, and CEACAM5 and that bind to domain 1 of CEACAM6. This is exemplified by the fact that blockade of CEACAM6 by antibodies of the invention increased IFN-γ secretion by >1.5-fold in co-culture of survivin-peptide-specific CD8 ⁺ T cells with KS tumor cells compared to control samples treated with isotype-matched controls ( Figures 5 and 6 ).

One embodiment of the present invention is to provide an antibody, or an antigen-binding antibody fragment, or a variant thereof, that specifically binds to human CEACAM6 and cynomolgus monkey CEACAM6 and is capable of altering the cytokine profile of tumor antigen-specific T cells toward a more cytotoxic and/or activated phenotype, wherein the more cytotoxic and/or activated phenotype is characterized by increased secretion of IFN-γ and/or IL-2 and/or TNF-α. Preferably, the tumor antigen-specific T cells are CD8 ⁺ T cells, and the phenotype is increased secretion of IFN-γ, IL-2, and TNF-α. If the antibody in a co-culture of tumor-specific CD8 ⁺ T cells with tumor cells produces an increase in IFN-γ and/or IL-2 and/or TNF-α secretion by >1.2-fold, preferably >1.5-fold, compared to a control sample treated with an isotype-matched control, then the anti-CEACAM6 antibody is capable of altering the cytokine profile of tumor antigen-specific T cells toward a more cytotoxic and/or activated phenotype. Blockade of CEACAM6 by antibodies of the invention resulted in >1.5-fold increase in IFN-γ, IL-2, and TNF-α secretion in co-culture of survivin-peptide-specific T cells with KS tumor cells compared to control samples treated with isotype-matched controls ( FIG6 ).

One embodiment of the present invention is to provide antibodies or antigen-binding antibody fragments or variants thereof that bind to a wide range of different cell lines expressing CEACAM6, including but not limited to the cell lines shown in the examples. These examples include human cell lines from a variety of tumor sources (e.g., NSCLC, SCLC, CRC, PancCA, breast CA, gastric CA, multiple myeloma, cervical cancer, skin cancer, which represent cancer indications that have been previously described in the literature as CEACAM6-positive (see introduction or review by Beauchimen and Arabzadeh, Cancer Metastasis Rev. 2013 Dec;32(3-4):643-71).

One embodiment of the present invention is to provide antibodies or antigen-binding antibody fragments or variants thereof that are safe for human administration. Preferably, the antibodies are chimeric, humanized or human. Very preferably, humanized antibodies or human antibodies.

Nevertheless, in certain assays it is preferred that the antibodies of the invention are expressed as murine IgG; for example, immunohistochemistry using human samples can be more readily analyzed by using murine antibodies or human-mouse chimeric antibodies.

Another embodiment of the present invention is to provide antibodies that constitute tools for diagnosing malignant or dysplastic conditions in which CEACAM6 expression is elevated compared to normal tissue or CEACAM6 is shed from the cell surface and becomes detectable in serum. Anti-CEACAM6 antibodies conjugated to a detectable label are provided. Preferred labels are radiolabels, enzymes, chromophores, or fluorophores.

Throughout this document, reference is made to the following preferred anti-CEACAM6 antibodies of the invention as described in Table 1.

Table 1: Protein sequences of preferred antibodies of the invention.

The sequences of the preferred antibodies or antigen-binding fragments thereof of the present invention shown in Table 1 are further provided and explained in FIG. 8 .

TPP-3308 represents an antibody comprising a heavy chain region corresponding to SEQ ID NO:43 and a light chain region corresponding to SEQ ID NO:44.

TPP-3310 represents an antibody comprising a heavy chain region corresponding to SEQ ID NO:57 and a light chain region corresponding to SEQ ID NO:58.

TPP-3714 represents an antibody comprising a heavy chain region corresponding to SEQ ID NO: 129 and a light chain region corresponding to SEQ ID NO: 130.

TPP-3323 represents an antibody comprising a heavy chain region corresponding to SEQ ID NO:85 and a light chain region corresponding to SEQ ID NO:86.

TPP-3820 represents an antibody comprising a heavy chain region corresponding to SEQ ID NO: 143 and a light chain region corresponding to SEQ ID NO: 144.

TPP-3821 represents an antibody comprising a heavy chain region corresponding to SEQ ID NO: 157 and a light chain region corresponding to SEQ ID NO: 158.

TPP-3322 represents an antibody comprising a heavy chain region corresponding to SEQ ID NO:71 and a light chain region corresponding to SEQ ID NO:72.

TPP-3707 represents an antibody comprising a heavy chain region corresponding to SEQ ID NO: 115 and a light chain region corresponding to SEQ ID NO: 116.

TPP-3705 represents an antibody comprising a heavy chain region corresponding to SEQ ID NO: 101 and a light chain region corresponding to SEQ ID NO: 102.

In a further preferred embodiment, the antibody or antigen-binding fragment comprises a heavy chain or light chain CDR sequence, and the heavy chain or light chain CDR sequence corresponds to at least one CDR sequence of the antibodies "TPP-2971", "TPP-3186", "TPP-3187", "TPP-3308", "TPP-3310", "TPP-3322", "TPP-3323", "TPP-3705", "TPP-3707", "TPP-3714", "TPP-3820", "TPP-3821", preferably the corresponding CDR sequence. The sequences are at least 50%, 55%, 60%, 70%, 80%, 90% or 95% identical, or at least 50%, 60%, 70%, 80%, 90%, 92% or 95% identical to the VH or VL sequence of "TPP-2971", "TPP-3186", "TPP-3187", "TPP-3308", "TPP-3310", "TPP-3322", "TPP-3323", "TPP-3705", "TPP-3707", "TPP-3714", "TPP-3820", "TPP-3821".

In a further preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises at least one CDR sequence or at least one variable heavy chain or variable light chain sequence as shown in Table 1.

In a preferred embodiment, an antibody or antigen-binding fragment thereof of the invention contains a heavy chain antigen-binding region comprising SEQ ID NO: 4 (H-CDR1), SEQ ID NO: 5 (H-CDR2), and SEQ ID NO: 6 (H-CDR3) and a light chain antigen-binding region comprising SEQ ID NO: 8 (L-CDR1), SEQ ID NO: 9 (L-CDR2), and SEQ ID NO: 10 (L-CDR3).

In a preferred embodiment, an antibody or antigen-binding fragment thereof of the invention contains a heavy chain antigen-binding region comprising SEQ ID NO: 34 (H-CDR1), SEQ ID NO: 35 (H-CDR2), and SEQ ID NO: 36 (H-CDR3) and a light chain antigen-binding region comprising SEQ ID NO: 38 (L-CDR1), SEQ ID NO: 39 (L-CDR2), and SEQ ID NO: 40 (L-CDR3).

In a preferred embodiment, an antibody or antigen-binding fragment thereof of the invention contains a heavy chain antigen-binding region comprising SEQ ID NO: 48 (H-CDR1), SEQ ID NO: 49 (H-CDR2), and SEQ ID NO: 50 (H-CDR3) and a light chain antigen-binding region comprising SEQ ID NO: 52 (L-CDR1), SEQ ID NO: 53 (L-CDR2), and SEQ ID NO: 54 (L-CDR3).

In a preferred embodiment, an antibody or antigen-binding fragment thereof of the invention contains a heavy chain antigen-binding region comprising SEQ ID NO: 120 (H-CDR1), SEQ ID NO: 121 (H-CDR2), and SEQ ID NO: 122 (H-CDR3) and a light chain antigen-binding region comprising SEQ ID NO: 124 (L-CDR1), SEQ ID NO: 125 (L-CDR2), and SEQ ID NO: 126 (L-CDR3).

In a preferred embodiment, an antibody or antigen-binding fragment thereof of the invention contains a heavy chain antigen-binding region comprising SEQ ID NO: 24 (H-CDR1), SEQ ID NO: 25 (H-CDR2), and SEQ ID NO: 26 (H-CDR3) and a light chain antigen-binding region comprising SEQ ID NO: 28 (L-CDR1), SEQ ID NO: 29 (L-CDR2), and SEQ ID NO: 30 (L-CDR3).

In a preferred embodiment, an antibody or antigen-binding fragment thereof of the invention contains a heavy chain antigen-binding region comprising SEQ ID NO: 76 (H-CDR1), SEQ ID NO: 77 (H-CDR2), and SEQ ID NO: 78 (H-CDR3) and a light chain antigen-binding region comprising SEQ ID NO: 80 (L-CDR1), SEQ ID NO: 81 (L-CDR2), and SEQ ID NO: 82 (L-CDR3).

In a preferred embodiment, an antibody or antigen-binding fragment thereof of the invention contains a heavy chain antigen-binding region comprising SEQ ID NO: 134 (H-CDR1), SEQ ID NO: 135 (H-CDR2), and SEQ ID NO: 136 (H-CDR3) and a light chain antigen-binding region comprising SEQ ID NO: 138 (L-CDR1), SEQ ID NO: 139 (L-CDR2), and SEQ ID NO: 140 (L-CDR3).

In a preferred embodiment, an antibody or antigen-binding fragment thereof of the invention contains a heavy chain antigen-binding region comprising SEQ ID NO: 148 (H-CDR1), SEQ ID NO: 149 (H-CDR2), and SEQ ID NO: 150 (H-CDR3) and a light chain antigen-binding region comprising SEQ ID NO: 152 (L-CDR1), SEQ ID NO: 153 (L-CDR2), and SEQ ID NO: 154 (L-CDR3).

In a preferred embodiment, an antibody or antigen-binding fragment thereof of the invention contains a heavy chain antigen-binding region comprising SEQ ID NO: 62 (H-CDR1), SEQ ID NO: 63 (H-CDR2), and SEQ ID NO: 64 (H-CDR3) and a light chain antigen-binding region comprising SEQ ID NO: 66 (L-CDR1), SEQ ID NO: 67 (L-CDR2), and SEQ ID NO: 68 (L-CDR3).

In a preferred embodiment, an antibody or antigen-binding fragment thereof of the invention contains a heavy chain antigen-binding region comprising SEQ ID NO: 14 (H-CDR1), SEQ ID NO: 15 (H-CDR2), and SEQ ID NO: 16 (H-CDR3) and a light chain antigen-binding region comprising SEQ ID NO: 18 (L-CDR1), SEQ ID NO: 19 (L-CDR2), and SEQ ID NO: 20 (L-CDR3).

In a preferred embodiment, an antibody or antigen-binding fragment thereof of the invention contains a heavy chain antigen-binding region comprising SEQ ID NO: 106 (H-CDR1), SEQ ID NO: 107 (H-CDR2), and SEQ ID NO: 108 (H-CDR3) and a light chain antigen-binding region comprising SEQ ID NO: 110 (L-CDR1), SEQ ID NO: 111 (L-CDR2), and SEQ ID NO: 112 (L-CDR3).

In a preferred embodiment, an antibody or antigen-binding fragment thereof of the invention contains a heavy chain antigen-binding region comprising SEQ ID NO: 92 (H-CDR1), SEQ ID NO: 93 (H-CDR2), and SEQ ID NO: 94 (H-CDR3) and a light chain antigen-binding region comprising SEQ ID NO: 96 (L-CDR1), SEQ ID NO: 97 (L-CDR2), and SEQ ID NO: 98 (L-CDR3).

Antibodies differ in sequence not only in their complementary determining regions (CDRs) but also in their frameworks (FRs). These sequence differences are encoded in different V-genes. The human antibody germline repertoire has been completely sequenced. There are approximately 50 functional VH germline genes, which can be divided into six subfamilies, VH1, VH2, VH3, VH4, VH5, and VH6, based on sequence homology (Tomlinson et al., 1992, J. Mol. Biol. 227, 776-798; Matsuda & Honjo, 1996, Advan. Immunol. 62, 1-29). About 40 functional VLκ genes are known to comprise seven subfamilies (Cox et al., 1994, Eur. J. Immunol. 24, 827-836; Barbie & Lefranc, 1998, Exp. Clin. Immunogenet. 15, 171-183), Vκ1, Vκ2, Vκ3, Vκ4, Vκ5, Vκ6 and Vκ7. Disclosed herein are the heavy chains of the antibodies of the present invention belonging to the human VH2 subfamily and the light chains of the antibodies of the present invention belonging to the human Vκ1 subfamily. It is known that the framework sequences of antibodies belonging to the same subfamily are closely related, for example, antibodies comprising members of the human VH3 subfamily all share considerable stability (Honegger et al., 2009, Protein Eng Des Sel. 22(3):121-134). It is well known in the art that CDRs from antibodies can be grafted onto different frameworks while maintaining the specific characteristics of the corresponding original antibodies. CDRs have been successfully grafted onto frameworks belonging to different species as well as onto frameworks of the same species belonging to different subfamilies. In a further embodiment, the antibody or antigen-binding fragment of the invention comprises at least one CDR sequence of an antibody of the invention and a human variable chain framework sequence as shown in Table 1.

In a preferred embodiment, the antibodies or antigen-binding fragments of the invention contain a variable light chain or light chain antigen-binding region comprising the L-CDR1, L-CDR2 and L-CDR3 sequences of the variable light chain and a variable heavy chain or heavy chain antigen-binding region comprising the H-CDR1, H-CDR2 and H-CDR3 sequences of the variable heavy chain of an antibody of the invention as shown in Table 1, as well as human variable light chain and human variable heavy chain framework sequences.

In a highly preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a variable heavy chain sequence (VH) as shown in SEQ ID NO:3 and a variable light chain sequence (VL) as shown in SEQ ID NO:7.

In a highly preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a variable heavy chain sequence (VH) as shown in SEQ ID NO:33 and a variable light chain sequence (VL) as shown in SEQ ID NO:37.

In a highly preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a variable heavy chain sequence (VH) as shown in SEQ ID NO:47 and a variable light chain sequence (VL) as shown in SEQ ID NO:51.

In a highly preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a variable heavy chain sequence (VH) as shown in SEQ ID NO:119 and a variable light chain sequence (VL) as shown in SEQ ID NO:123.

In a highly preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a variable heavy chain sequence (VH) as shown in SEQ ID NO:23 and a variable light chain sequence (VL) as shown in SEQ ID NO:27.

In a highly preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a variable heavy chain sequence (VH) as shown in SEQ ID NO:75 and a variable light chain sequence (VL) as shown in SEQ ID NO:79.

In a highly preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a variable heavy chain sequence (VH) as shown in SEQ ID NO:133 and a variable light chain sequence (VL) as shown in SEQ ID NO:137.

In a highly preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a variable heavy chain sequence (VH) as shown in SEQ ID NO:147 and a variable light chain sequence (VL) as shown in SEQ ID NO:151.

In a highly preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a variable heavy chain sequence (VH) as shown in SEQ ID NO:13 and a variable light chain sequence (VL) as shown in SEQ ID NO:17.

In a highly preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a variable heavy chain sequence (VH) as shown in SEQ ID NO:61 and a variable light chain sequence (VL) as shown in SEQ ID NO:65.

In a highly preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a variable heavy chain sequence (VH) as shown in SEQ ID NO:105 and a variable light chain sequence (VL) as shown in SEQ ID NO:109.

In a highly preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a variable heavy chain sequence (VH) as shown in SEQ ID NO:91 and a variable light chain sequence (VL) as shown in SEQ ID NO:95.

In some embodiments, the anti-CEACAM6 antibody or antigen-binding antibody fragment or variant thereof of the present invention binds to an epitope of human CEACAM6, wherein the epitope comprises one or more amino acid residues selected from Gln60, Asn61, Arg62, Ile63, Val83, Ile84, Gly85, Thr90, Ser127, Asp128 and Leu129 of SEQ ID NO: 179.

In certain embodiments, the anti-CEACAM6 antibodies or antigen-binding antibody fragments or variants thereof of the present invention bind to an epitope of human CEACAM6, wherein the epitope comprises amino acid residues Gln60, Asn61, Arg62, Ile63, Val83, Ile84, Gly85, Thr90, Ser127, Asp128 and Leu129 of SEQ ID NO: 179.

In some embodiments, the anti-CEACAM6 antibodies or antigen-binding antibody fragments or variants thereof of the present invention interact with, e.g., bind to, an epitope of human CEACAM6, wherein the epitope comprises one, two, three, four, five, eight, ten, fifteen or more amino acid residues selected from Pro59, Gln60, Asn61, Arg62, Ile63, Gly64, Val83, Ile84, Gly85, Thr86, Gln88, Thr90, Pro91, Ile125, Ser127, Asp128 and Leu129 of SEQ ID NO: 179.

In certain embodiments, the anti-CEACAM6 antibodies, or antigen-binding antibody fragments or variants thereof, of the present invention interact with, e.g., bind to, an epitope of human CEACAM6, wherein the epitope comprises amino acid residues Pro59, Gln60, Asn61, Arg62, Ile63, Gly64, Val83, Ile84, Gly85, Thr86, Gln88, Thr90, Pro91, Ile125, Ser127, Asp128, and Leu129 of SEQ ID NO: 179.

In some embodiments, the anti-CEACAM6 antibody, or antigen-binding antibody fragment, or variant thereof of the present invention binds to an epitope of human CEACAM6, wherein the epitope comprises one or more amino acid residues selected from Gln60, Asn61, Arg62, Ile63, Val83, Ile84, Gly85, Thr90, Ser127, Asp128, and Leu129 of SEQ ID NO: 179, and wherein the antibody, or antigen-binding antibody fragment, or variant thereof binds to a human CEACAM6 protein comprising an Ile63Leu mutation, and does not bind to a human CEACAM6 protein comprising an Ile63Phe mutation (numbered according to SEQ ID NO: 179).

In certain embodiments, the anti-CEACAM6 antibody, or antigen-binding antibody fragment, or variant thereof of the present invention binds to an epitope of human CEACAM6, wherein the epitope comprises amino acid residues Gln60, Asn61, Arg62, Ile63, Val83, Ile84, Gly85, Thr90, Ser127, Asp128, and Leu129 of SEQ ID NO: 179, and wherein the antibody, or antigen-binding antibody fragment, or variant thereof binds to a human CEACAM6 protein comprising an Ile63Leu mutation and does not bind to a human CEACAM6 protein comprising an Ile63Phe mutation (numbered according to SEQ ID NO: 179).

In some embodiments, the anti-CEACAM6 antibody, or antigen-binding antibody fragment, or variant thereof of the present invention interacts with, e.g., binds to, an epitope of human CEACAM6, wherein the epitope comprises one, two, three, four, five, eight, ten, fifteen, or more amino acid residues selected from Pro59, Gln60, Asn61, Arg62, Ile63, Gly64, Val83, Ile84, Gly85, Thr86, Gln88, Thr90, Pro91, Ile125, Ser127, Asp128, and Leu129 of SEQ ID NO: 179, and wherein the antibody, or antigen-binding antibody fragment, or variant thereof, binds to a human CEACAM6 protein comprising an Ile63Leu mutation and does not bind to a human CEACAM6 protein comprising an Ile63Phe mutation (numbered according to SEQ ID NO: 179).

In certain embodiments, the anti-CEACAM6 antibodies, or antigen-binding antibody fragments or variants thereof, of the present invention interact with, e.g., bind to, an epitope of human CEACAM6, wherein the epitope comprises amino acid residues Pro59, Gln60, Asn61, Arg62, Ile63, Gly64, Val83, Ile84, Gly85, Thr86, Gln88, Thr90, Pro91, Ile125, Ser127, Aspl28, and Leu129 of SEQ ID NO: 179, and wherein the antibodies, or antigen-binding antibody fragments or variants thereof, bind to a human CEACAM6 protein comprising an Ile63Leu mutation and do not bind to a human CEACAM6 protein comprising an Ile63Phe mutation (numbered according to SEQ ID NO: 179).

In some embodiments, the anti-CEACAM6 antibody, or antigen-binding antibody fragment, or variant thereof of the present invention binds to an epitope of human CEACAM6, wherein the epitope comprises one or more amino acid residues selected from Gln60, Asn61, Arg62, Ile63, Val83, Ile84, Gly85, Thr90, Ser127, Asp128, and Leu129 of SEQ ID NO: 179, and wherein the antibody, or antigen-binding antibody fragment, or variant thereof competes for CEACAM6 binding with an antibody comprising a variable heavy chain sequence (VH) as shown in SEQ ID NO: 47 and a variable light chain sequence (VL) as shown in SEQ ID NO: 51.

In certain embodiments, the anti-CEACAM6 antibodies of the present invention bind to an epitope of human CEACAM6, wherein the epitope comprises amino acid residues Gln60, Asn61, Arg62, Ile63, Val83, Ile84, Gly85, Thr90, Ser127, Asp128 and Leu129 of SEQ ID NO: 179, and wherein the antibody or antigen-binding antibody fragment or variant thereof competes for binding to CEACAM6 with an antibody comprising a variable heavy chain sequence (VH) as shown in SEQ ID NO: 47 and a variable light chain sequence (VL) as shown in SEQ ID NO: 51.

In some embodiments, the anti-CEACAM6 antibodies of the present invention interact with, e.g., bind to, an epitope of human CEACAM6, wherein the epitope comprises one, two, three, four, five, eight, ten, fifteen or more amino acid residues selected from Pro59, Gln60, Asn61, Arg62, Ile63, Gly64, Val83, Ile84, Gly85, Thr86, Gln88, Thr90, Pro91, Ile125, Ser127, Asp128 and Leu129 of SEQ ID NO: 179, and wherein the antibody, or antigen-binding antibody fragment thereof, or variant thereof, competes for binding to CEACAM6 with an antibody comprising a variable heavy chain sequence (VH) as set forth in SEQ ID NO: 47 and a variable light chain sequence (VL) as set forth in SEQ ID NO: 51.

In certain embodiments, the anti-CEACAM6 antibodies of the present invention interact with, e.g., bind to, an epitope of human CEACAM6, wherein the epitope comprises amino acid residues Pro59, Gln60, Asn61, Arg62, Ile63, Gly64, Val83, Ile84, Gly85, Thr86, Gln88, Thr90, Pro91, Ile125, Ser127, Asp128, and Leu129 of SEQ ID NO: 179, and wherein the antibody, or antigen-binding antibody fragment thereof, or variant thereof, competes for binding to CEACAM6 with an antibody comprising a variable heavy chain sequence (VH) as set forth in SEQ ID NO: 47 and a variable light chain sequence (VL) as set forth in SEQ ID NO: 51.

In some embodiments, the anti-CEACAM6 antibody, or antigen-binding antibody fragment, or variant thereof of the present invention binds to an epitope of human CEACAM6, wherein the epitope comprises one or more amino acid residues selected from Gln60, Asn61, Arg62, Ile63, Val83, Ile84, Gly85, Thr90, Ser127, Asp128, and Leu129 of SEQ ID NO: 179, and wherein the antibody, or antigen-binding antibody fragment, or variant thereof competes for CEACAM6 binding with an antibody comprising a variable heavy chain sequence (VH) as shown in SEQ ID NO: 47 and a variable light chain sequence (VL) as shown in SEQ ID NO: 51, and wherein the antibody, or antigen-binding antibody fragment, or variant thereof binds to a human CEACAM6 protein comprising an Ile63Leu mutation and does not bind to a human CEACAM6 protein comprising an Ile63Phe mutation (numbered according to SEQ ID: 179).

In certain embodiments, the anti-CEACAM6 antibody of the present invention binds to an epitope of human CEACAM6, wherein the epitope comprises amino acid residues Gln60, Asn61, Arg62, Ile63, Val83, Ile84, Gly85, Thr90, Ser127, Asp128 and Leu129 of SEQ ID NO: 179, and wherein the antibody, or antigen-binding antibody fragment thereof, or variant thereof competes for binding to CEACAM6 with an antibody comprising a variable heavy chain sequence (VH) as shown in SEQ ID NO: 47 and a variable light chain sequence (VL) as shown in SEQ ID NO: 51, and wherein the antibody, or antigen-binding antibody fragment thereof, or variant thereof binds to a human CEACAM6 protein comprising an Ile63Leu mutation and does not bind to a human CEACAM6 protein comprising an Ile63Phe mutation (numbered according to SEQ-ID: 179).

In some embodiments, the anti-CEACAM6 antibodies of the present invention interact with, e.g., bind to, an epitope of human CEACAM6, wherein the epitope comprises one, two, three, four, five, eight, ten, fifteen, or more amino acid residues selected from Pro59, Gln60, Asn61, Arg62, Ile63, Gly64, Val83, Ile84, Gly85, Thr86, Gln88, Thr90, Pro91, Ile125, Ser127, Asp128, and Leu129 of SEQ ID NO: 179, and wherein the antibody, or antigen-binding antibody fragment thereof, or variant thereof, interacts with, e.g., a variable heavy chain sequence (VH) as set forth in SEQ ID NO: 47 and a variable heavy chain sequence (VH) as set forth in SEQ ID NO: 48. An antibody with the variable light chain sequence (VL) shown in NO:51 competes for binding to CECACAM6, wherein the antibody or its antigen-binding antibody fragment or variant thereof binds to a human CEACAM6 protein comprising an Ile63Leu mutation and does not bind to a human CEACAM6 protein comprising an Ile63Phe mutation (numbered according to SEQ-ID:179).

In certain embodiments, the anti-CEACAM6 antibodies of the present invention interact with, e.g., bind to, an epitope of human CEACAM6, wherein the epitope comprises amino acid residues Pro59, Gln60, Asn61, Arg62, Ile63, Gly64, Val83, Ile84, Gly85, Thr86, Gln88, Thr90, Pro91, Ile125, Ser127, Asp128, and Leu129 of SEQ ID NO: 179, and wherein the antibody, or antigen-binding antibody fragment, or variant thereof, competes for CEACAM6 binding with an antibody comprising a variable heavy chain sequence (VH) as set forth in SEQ ID NO: 47 and a variable light chain sequence (VL) as set forth in SEQ ID NO: 51, and wherein the antibody, or antigen-binding antibody fragment, or variant thereof, binds to a human CEACAM6 protein comprising an Ile63Leu mutation and does not bind to a human CEACAM6 protein comprising an Ile63Phe mutation (numbered according to SEQ ID: 179).

The antibodies of the present invention may be IgG (immunoglobulin G, such as IgG1, IgG2, IgG3, IgG4) or IgA, IgD, IgE, IgM, and the antibody fragments may be, for example, Fab, Fab', F(ab') ₂ , Fab'-SH or scFv. Thus, the antibody fragments of the present invention may be or may contain an antigen binding region that functions in one or more of the ways described herein.

In a preferred embodiment, the antibodies or antigen-binding antibody fragments of the invention are monoclonal.

In some embodiments, the antibody of the present invention or its Fab or the nucleic acid encoding it is separated. The biological component of separation (such as nucleic acid molecules or proteins such as antibodies) is a biological component that has been substantially separated or purified from other biological components (such as other chromosomes and extrachromosomal DNA and RNA, proteins and organelles) in the cell of the organism in which the component is naturally present. The term also includes nucleic acids and proteins prepared by recombinant expression and chemically synthesized nucleic acids in host cells.

Antibody production

The antibodies of the present invention can be derived from recombinant antibody libraries based on amino acid sequences that have been isolated from antibodies of a large number of healthy volunteers, for example, using n-CoDeR® technology to reconstitute fully human CDRs into new antibody molecules (Carlson & Söderlind, Expert Rev Mol Diagn. 2001 May;1(1):102-8). Alternatively, antibody libraries that are fully human antibody phage display libraries, such as those described in Hoet RM et al., Nat Biotechnol 2005;23(3):344-8), can be used to isolate CEACAM6-specific antibodies. Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

Human antibodies can be further prepared by administering an immunogen to a transgenic animal that has been modified to produce a complete human antibody or a complete antibody having a human variable region in response to an antigenic challenge. Such animals typically contain all or a portion of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus, or that exists extrachromosomally or is randomly integrated into the chromosome of the animal. For example, immunization of genetically engineered mice, particularly hMAb mice (e.g., VelocImmune mice® or XENOMOUSE®) can be performed.

Further antibodies can be generated using hybridoma technology (see, eg, Köhler and Milstein Nature. 1975 Aug 7; 256(5517):495-7) resulting in, eg, murine, rat or rabbit antibodies, which can be converted into chimeric or humanized antibodies. Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Natl Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osboum et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the "guided selection" method for FR shuffling).

An example of the use of recombinant antibody libraries to generate antibodies in combination with mouse immunization and subsequent humanization is provided.

A further aspect of the present invention is to provide a method for generating antibodies that specifically bind to human CEACAM6 and cynomolgus monkey CEACAM6 and do not significantly cross-react with human CEACAM1, human CEACAM3, and human CEACAM5. One embodiment of the present invention is to provide a method for generating anti-CEACAM6 antibodies, characterized by comprising the steps of immunizing an animal, preferably a mouse, with cynomolgus monkey CECAM6 domain 1 (represented by amino acids 35-142 of SEQ-ID NO: 177), determining the amino acid sequence of an antibody that specifically binds to human CEACAM6 and cynomolgus monkey CEACAM6, and then optionally humanizing or generating a chimeric antibody and expressing the antibody. The expression system can be a recombinant or cell-free expression system. Suitable host cells for recombinant expression are prokaryotic and eukaryotic cells. Mammalian expression systems are preferred.

Peptide variants

The antibodies or antigen-binding fragments of the present invention are not limited to the specific peptide sequences provided herein. Rather, the present invention also encompasses variants of these polypeptides. Given this disclosure and conventionally available techniques and references, one of skill will be able to prepare, test, and utilize functional variants of the antibodies disclosed herein, understanding that such variants possess the ability to bind to CEACAM6 fall within the scope of the present invention.

Variants can include, for example, antibodies having at least one altered complementarity determining region (CDR) (hypervariable) and/or framework (FR) (variable) domain/position relative to the peptide sequences disclosed herein.

By altering one or more amino acid residues in the CDR or FR regions, one can routinely generate mutated or diversified antibody sequences, which can be screened for new or improved properties against the antigen, for example.

A further preferred embodiment of the present invention is an antibody or antigen-binding fragment in which the VH and VL sequences are selected as shown in Table 1. A skilled artisan can use the data in Table 1 to design peptide variants within the scope of the present invention. Variants are preferably constructed by altering amino acids within one or more CDR regions; variants may also have one or more altered framework regions. Alterations may also be made within the framework regions. For example, peptide FR domains may be altered in which residues deviate from the germline sequence.

Alternatively, one can perform the same analysis by comparing the amino acid sequences disclosed herein with known sequences of the same type of such antibodies using, for example, the program described in Knappik A., et al., JMB 2000, 296:57-86.

In addition, variants can be obtained by: using a kind of antibody as the starting point for further optimization, the further optimization is by making one or more amino acid residues in the antibody, preferably making the amino acid residue in one or more CDRs diversify, and for the variant screening obtained antibody variant set with improved properties. Particularly preferred is the diversification of one or more amino acid residues in the CDR3 of VL and/or VH. Diversification can be carried out, for example, by using the set of trinucleotide mutagenesis (TRIM) technology synthetic DNA molecules (Virnekäs B. et al., Nucl. Acids Res. 1994, 22: 5600.). Antibody or its antigen-binding fragment include molecules with modification/variation, including but not limited to, for example, causing half-life changes (such as modification of Fc part or connection of other molecules such as PEG), binding affinity changes or ADCC or CDC activity changes.

Conservative amino acid variants

Polypeptide variants can be prepared that retain the overall molecular structure of the antibody peptide sequences described herein. In view of the properties of individual amino acids, the skilled artisan will recognize some reasonable substitutions. For example, amino acid substitutions can be made based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic properties of the residues, i.e., "conservative substitutions."

For example, (a) non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; (b) polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positively charged (basic) amino acids include arginine, lysine, and histidine; and (d) negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Substitutions are generally made within groups (a)-(d). In addition, glycine and proline can be substituted for each other based on their ability to disrupt α-helices. Similarly, certain amino acids, such as alanine, cysteine, leucine, methionine, glutamic acid, glutamine, histidine, and lysine, are more commonly found in α-helices, while valine, isoleucine, phenylalanine, tyrosine, tryptophan, and threonine are more commonly found in β-pleated sheets. Glycine, serine, aspartic acid, asparagine, and proline are commonly found in turns. Some preferred substitutions can be made in the following groups: (i) S and T; (ii) P and G; and (iii) A, V, L, and I. Given the known genetic code and recombinant and synthetic DNA technology, skilled researchers can readily construct DNA encoding conservative amino acid variants.

Glycosylation variants

When the antibody comprises an Fc region, the carbohydrate attached to the Fc region can be altered. Natural antibodies produced by mammalian cells typically contain branched, bibranched oligosaccharides, which are typically attached to Asn297 of the CH2 domain of the Fc region via an N-linkage (using Kabat EU numbering); see, e.g., Wright et al. Trends Biotechnol. 15: 26-32 (1997).

In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree to which the antibodies are glycosylated. Adding or deleting glycosylation sites to antibodies can be easily accomplished by changing the expression system (e.g., host cell) and/or by changing the amino acid sequence, such that one or more glycosylation sites are generated or removed.

In one embodiment of the present invention, aglycosyl antibodies or antibody derivatives with reduced effector function are prepared by expression in prokaryotic hosts. Suitable prokaryotic hosts include, but are not limited to, Escherichia coli, Bacillus subtilis , Salmonella typhimurium , and various species of Pseudomonas, Streptomyces, and Staphylococcus.

In one embodiment, an antibody variant with reduced effector function is provided, characterized in that a modification at a conserved N-linked site in the CH2 domain of the Fc portion of the antibody is provided. In one embodiment of the invention, the modification comprises a mutation at a heavy chain glycosylation site to prevent glycosylation at the site. Therefore, in a preferred embodiment of the invention, an aglycosyl antibody or antibody derivative is prepared by a mutation of a heavy chain glycosylation site, i.e., a mutation of N297 using Kabat EU numbering, and expressed in an appropriate host cell.

In another embodiment of the present invention, the aglycosyl antibody or antibody derivative has reduced effector function, wherein the modification of the conserved N-linked site in the CH2 domain of the Fc portion of the antibody or antibody derivative comprises removal of CH2 domain glycans, i.e., deglycosylation. These aglycosyl antibodies can be produced by conventional methods and then enzymatically deglycosylated. Methods for enzymatic deglycosylation of antibodies are well known in the art (e.g., Winkelhake & Nicolson (1976), J Biol Chem. 251(4):1074-80).

In another embodiment of the present invention, deglycosylation can be achieved using the glycosylation inhibitor tunicamycin (Nose & Wigzell (1983), Proc Natl Acad Sci USA, 80(21):6632-6). In other words, the modification is to prevent glycosylation at the conserved N-linked site in the CH2 domain of the Fc portion of the antibody.

In one embodiment, antibody variants are provided that lack carbohydrate structures that are fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all sugar structures attached to Asn297 as measured by MALDI-TOF mass spectrometry (e.g., complex, hybrid, and high mannose structures), as described, for example, in WO 2008/077546. Asn297 refers to the asparagine residue located at approximately position 297 (EU numbering of Fc region residues) in the Fc region; however, due to minor sequence variations in antibodies, Asn297 may also be located approximately ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function.

Disclosed examples of "defucosylated" or "fucose-deficient" antibody variants include: Okazaki et al. J Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).

Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); and WO 2004/056312) and knockout cell lines, such as CHO cells in which the α-1,6-fucosyltransferase gene FUT8 is knocked out (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006)).

Also provided are antibody variants having bisected oligosaccharides, e.g., bisected oligosaccharides attached to the Fc region of the antibody by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described in, e.g., WO 2003/011878; U.S. Patent No. 6,602,684; and US 2005/0123546.

Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087; WO 1998/58964; and WO 1999/22764.

Fc region variants

In certain embodiments, one or more amino acid modifications (eg, substitutions) can be introduced into the Fc region of an antibody provided herein (eg, a human IgG1, IgG2, IgG3, or IgG4 Fc region), thereby generating an Fc region variant.

In certain embodiments, the present invention contemplates antibody variants that possess some, but not all, effector functions, making them desirable candidates for applications where the in vivo half-life of the antibody is important but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays can be performed to confirm reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity) but retains FcRn binding ability. In some embodiments, changes are made in the Fc region that result in altered (i.e., improved or reduced) C1q binding and/or complement dependent cytotoxicity (CDC).

In certain embodiments, the present invention contemplates antibody variants with increased or decreased half-life. Antibodies with increased half-life and improved binding to neonatal Fc receptors (FcRn) that are responsible for transferring maternal IgG to the fetus (Guyer et al., J Immunol. 117: 587 (1976) and Kim et al., J Immunol. 24: 249 (1994)) are described in US 2005/0014934 (Hinton et al.). Those antibodies comprise Fc regions with one or more substitutions that improve the binding of the Fc regions to FcRn.

Antibody-drug conjugates (ADCs)

The present invention also provides antibody-drug conjugates (ADCs, immunoconjugates) comprising an anti-CEACAM6 antibody conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins of bacterial, fungal, plant or animal origin, enzymatically active toxins or fragments thereof), or radioactive isotopes.

In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0425235B1); auristatins, such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin; calicheamicin or a derivative thereof; anthracyclines, such as daunorubicin or doxorubicin; methotrexate; vindesine; taxanes, such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortaxel; trichothecenes; and CC1065.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, a nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (P API, P APII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis toxin, scutellaria baicalensis toxin, scutellaria serrata ... officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and tricothecenes.

In another embodiment, the immunoconjugate comprises an antibody as described herein that is conjugated to a radioactive atom to form a radioconjugate. A variety ^of radioisotopes can be used to produce radioconjugates. Examples include ^Th , Ac, At, ^I , ^I , Y ^{, Re , Re} , ^Sm , ^Bi , ^P , Pb, and ^{radioisotopes} of Lu. When the radioconjugate is used for detection, it can include radioactive atoms for scintigraphic studies, such as Tc99m, or spin labels for nuclear magnetic resonance (NMR) imaging, such as iodine-123 (again), iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

Conjugates of the antibody and cytotoxic agent can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl diimidoadipate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

The linker can be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell, for example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Res. 52: 127-131 (1992)).

The immunoconjugates or ADCs herein specifically contemplate, but are not limited to, such conjugates prepared with cross-linkers including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which is commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A.).

DNA molecules of the present invention

The present invention further relates to the DNA molecules encoding the antibodies of the present invention or their antigen-binding fragments. The DNA sequences of the antibodies for expression are provided in Table 32. These sequences are optimized for mammalian expression in some cases. The DNA molecules of the present invention are not limited to the sequences disclosed herein, but also include variants thereof. DNA variants within the present invention can be described by reference to their physical properties in hybridization. Technicians will recognize that nucleic acid hybridization techniques can be used to identify their complementary sequences (because DNA is double-stranded) and their equivalents or homologues. It will also be recognized that hybridization can be performed with less than 100% complementarity. However, in view of appropriate condition selection, hybridization techniques can be used to distinguish DNA sequences based on the structural correlation of DNA sequences and specific probes. For guidance on such conditions, see Sambrook et al., 1989 supra and Ausubel et al., 1995 (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Sedman, J. G., Smith, J. A., & Struhl, K. eds. (1995). Current Protocols in Molecular Biology. New York: John Wiley and Sons).

The structural similarity between two polynucleotide sequences can be expressed as a function of the "stringency" of the conditions under which the two sequences will hybridize to each other. As used herein, the term "stringency" refers to the degree to which the conditions are unfavorable for hybridization. Stringent conditions strongly discourage hybridization, and under such conditions, only the most structurally related molecules will hybridize to each other. In contrast, non-stringent conditions favor the hybridization of molecules that exhibit a lower degree of structural relatedness. Therefore, hybridization stringency is directly related to the structural relationship of the two nucleic acid sequences.

Hybridization stringency varies with many factors, including total DNA concentration, ionic strength, temperature, probe size, and the presence of agents that disrupt hydrogen bonding. Factors that promote hybridization include high DNA concentration, high ionic strength, low temperature, longer probe size, and the absence of agents that disrupt hydrogen bonding. Hybridization is typically carried out in two stages: a "binding" stage and a "washing" stage.

Functionally equivalent DNA variants

Yet another class of DNA variants within the scope of the present invention may be described by reference to the product they encode.These functionally equivalent polynucleotides are characterized by the fact that, due to the degeneracy of the genetic code, they encode the same peptide sequence.

It should be appreciated that variants of the DNA molecules provided herein can be constructed in several different ways. For example, they can be constructed as fully synthetic DNA. Methods for effectively synthesizing oligonucleotides are widely available. See Ausubel et al., Section 2.11, Supplementary Material 21 (1993). Overlapping oligonucleotides can be synthesized and assembled in a manner first reported by Khorana et al ., J. Mol. Biol. 72:209-217 (1971); see also Ausubel et al., supra, Section 8.2. Preferably, the synthetic DNA is designed with convenient restriction sites engineered at the 5' and 3' ends of the gene to facilitate cloning into an appropriate vector.

As shown, the method for generating variants starts with one of the DNA disclosed herein and then constructs site-directed mutagenesis. See Ausubel et al., supra , Chapter 8, Supplementary Material 37 (1997). In a typical method, the target DNA is cloned into a single-stranded DNA phage vector. The single-stranded DNA is isolated and hybridized with an oligonucleotide containing one or more desired nucleotide variations. The complementary strand is synthesized and the double-stranded phage is introduced into the host. Some of the resulting progeny will contain the desired mutant, which can be confirmed using DNA sequencing. In addition, various methods that increase the probability of progeny phage becoming a desired mutant are available. These methods are well known to those skilled in the art, and the kit for generating such mutants is commercially available.

Recombinant DNA constructs and expression

The present invention further provides recombinant DNA constructs comprising one or more of the nucleotide sequences of the present invention (see Table 32). The recombinant constructs of the present invention can be used in conjunction with vectors (such as plasmids, phagemids, phage or viral vectors) into which a DNA molecule encoding an antibody or antigen-binding fragment thereof of the present invention or a variant thereof is inserted.

Antibodies, antigen-binding portions thereof, or variants provided herein can be prepared by recombinantly expressing nucleic acid sequences encoding light and heavy chains or portions thereof in host cells. In order to recombinantly express antibodies, antigen-binding portions thereof, or variants, host cells can be transfected with one or more recombinant expression vectors (which carry DNA fragments encoding light and/or heavy chains or portions thereof) so that light and heavy chains are expressed in host cells. Nucleic acids encoding heavy and light chains are prepared and/or obtained using standard recombinant DNA methods, these nucleic acids are incorporated into recombinant expression vectors, and the vectors are introduced into host cells, such as those described in: Sambrook, Fritsch and Maniatis (ed.), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F. M. et al. (ed.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and U.S. Patent No. 4,816,397 by Boss et al.

In addition, the nucleic acid sequence encoding the variable region of the heavy chain and/or light chain can be converted into a nucleic acid sequence encoding, for example, a full-length antibody chain, a Fab fragment or an scFv. The DNA fragment encoding VL or VH can be operably linked to another DNA fragment encoding, for example, an antibody constant region or a flexible linker (such that the amino acid sequence encoded by the two DNA fragments is in frame). The sequences of human heavy and light chain constant regions are known in the art (see, for example, Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification.

To generate a polynucleotide sequence encoding an scFv, nucleic acids encoding VH and VL can be operably linked to another segment encoding a flexible linker so that the VH and VL sequences can be expressed as a contiguous single-chain protein in which the VL and VH regions are connected by a flexible linker (see, e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 348:552-554).

In order to express antibodies, their antigen-binding fragments or variants thereof, standard recombinant DNA expression methods can be used (see, for example, Goeddel; Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). For example, DNA encoding the desired polypeptide can be inserted into an expression vector and then transfected into a suitable host cell. Suitable host cells are prokaryotic cells and eukaryotic cells. Examples of prokaryotic host cells are, for example, bacteria, and examples of eukaryotic host cells are yeast, insects and insect cells, plants and plant cells, transgenic animals or mammalian cells. In some embodiments, the DNA encoding the heavy chain and light chain are inserted into different vectors. In other embodiments, the DNA encoding the heavy chain and light chain are inserted into the same vector. It should be understood that the design of the expression vector, including the selection of regulatory sequences, is affected by factors such as, for example, the choice of host cells, the expression level of the desired protein, and whether the expression is constitutive or inducible.

Thus, one embodiment of the present invention is also a host cell comprising a vector or nucleic acid molecule, whereby the host cell may be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, and may be a prokaryotic cell, such as a bacterial cell.

Another embodiment of the present invention is a method of using host cells to produce antibodies and antigen-binding fragments, which comprises culturing the host cells under suitable conditions and recovering the antibodies.

Therefore, another embodiment of the present invention is to use the host cells of the present invention to produce antibodies according to the present invention and to purify these antibodies to at least 95% homogeneity by weight.

Bacterial expression

Useful expression vectors for bacterial applications are constructed by inserting the DNA sequence encoding the desired protein together with suitable translation initiation and termination signals in an operable reading phase with a functional promoter. The vector may comprise one or more phenotypic selection markers and origins of replication to ensure maintenance of the vector and, if necessary, to provide amplification in the host. Suitable prokaryotic hosts for transformation include, but are not limited to, Escherichia coli ( E. coli ), Bacillus subtilis ( Bacillus subtilis ), Salmonella typhimurium (Salmonella typhimurium ) and each species within Pseudomonas (Pseudomonas), Streptomyces (Streptomyces) and Staphylococcus (Staphylococcus).

Bacterial vectors can be, for example, based on phage, plasmid or phagemid. These vectors can contain selective markers and the bacterial replication origin that is derived from the plasmid available in commerce, and the plasmid usually contains the element of well-known cloning vector pBR322 (ATCC 37017). After suitable host strain and host strain are grown to appropriate cell density, by appropriate means (such as temperature variation or chemical induction) repression/induction selected promotor is removed, and cell is cultivated for a period of time. Cell is usually gathered in the crops by centrifugation, destroyed by physical or chemical means, and retains gained crude extract for further purification.

In bacterial systems, a variety of expression vectors can be advantageously selected depending on the intended use of the expressed protein. For example, when large quantities of such proteins are to be produced for use, for example, in generating antibodies or screening peptide libraries, vectors that direct the expression of high-level fusion protein products that are easily purified may be desirable.

Thus, one embodiment of the present invention is an expression vector comprising a nucleic acid sequence encoding a novel antibody of the present invention.

The antibodies or antigen-binding fragments thereof or variants thereof of the present invention include naturally purified products, products of chemical synthesis procedures, and products produced by recombinant technology from the following prokaryotic hosts, including, for example, Escherichia coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, preferably products produced from Escherichia coli cells.

Mammalian expression

Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high-level expression of proteins in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter/enhancer), simian virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), and polyoma virus. Expression of the antibody can be constitutive or regulated (e.g., by addition or removal of small molecule inducers such as tetracycline in combination with the Tet system). For further description of viral regulatory elements and their sequences, see, for example, U.S. 5,168,062 to Stinski, U.S. 4,510,245 to Bell et al., and U.S. 4,968,615 to Schaffner et al. The recombinant expression vector may also include an origin of replication and a selectable marker (see, for example, U.S. 4,399,216, 4,634,665, and U.S. 5,179,017). Suitable selectable markers include genes that confer resistance to drugs (such as G418, puromycin, hygromycin, blasticidin, zeocin/bleomycin, or methotrexate) on the host cell into which the vector has been introduced, or selectable markers utilizing auxotrophy, such as glutamine synthetase (Bebbington et al., Biotechnology (NY). 1992 Feb; 10(2): 169-75). For example, the dihydrofolate reductase (DHFR) gene confers resistance to methotrexate, the neo gene confers resistance to G418, the bsd gene from Aspergillus terreus confers resistance to blasticidin, puromycin N-acetyltransferase confers resistance to puromycin, the Sh ble gene product confers resistance to bleomycin, and resistance to hygromycin is conferred by the Escherichia coli hygromycin resistance gene (hyg or hph). Selectable markers such as DHFR or glutamine synthetase can also be used in amplification techniques combined with MTX and MSX.

Transfection of expression vectors into host cells can be performed using standard techniques such as electroporation, nucleofection, calcium phosphate precipitation, lipofection, polycation-based transfection such as polyethyleneimine (PEI)-based transfection, and DEAE-dextran transfection.

Suitable mammalian host cells for expressing the antibodies, antigen-binding fragments thereof, or variants thereof provided herein include Chinese hamster ovary (CHO cells) such as CHO-K1, CHO-S, CHO-K1SV [including dhfr-CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220 and Urlaub et al., Cell. 1983 Jun;33(2):405-12, used with, for example, the DHFR selection marker described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621; and Fan et al., Biotechnol Bioeng. 2012]. Apr; 109(4): 1007-15], NS0 myeloma cells, COS cells, HEK293 cells, HKB11 cells, BHK21 cells, CAP cells, EB66 cells and SP2 cells.

Expression may also be transient or semi-stable in expression systems such as HEK293, HEK293T, HEK293-EBNA, HEK293E, HEK293-6E, HEK293-Freestyle, HKB11, Expi293F, 293EBNALT75, CHO Freestyle, CHO-S, CHO-K1, CHO-K1SV, CHOEBNALT85, CHOS-XE, CHO-3E7, or CAP-T cells (e.g., Durocher et al., Nucleic Acids Res. 2002 Jan 15;30(2):E9).

In some embodiments, the expression vector is designed so that the expressed protein is secreted into the culture medium in which the host cells are grown. The antibody, antigen-binding fragment thereof, or variant thereof can be recovered from the culture medium using standard protein purification methods.

purification

The antibodies of the present invention or their antigen-binding fragments or variants thereof can be recovered and purified from recombinant cell cultures by well-known methods, including but not limited to ammonium sulfate or ethanol precipitation, acid extraction, protein A chromatography, protein G chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification. See, for example, Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each incorporated herein by reference in its entirety.

The antibodies or antigen-binding fragments thereof or variants thereof of the present invention include naturally purified products, products of chemical synthesis procedures, and products produced by recombinant technology from eukaryotic hosts (including, for example, yeast, higher plants, insects, and mammalian cells). Depending on the host utilized in the recombinant production procedure, the antibodies of the present invention may be glycosylated or may be non-glycosylated. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, sections 17.37-17.42; Ausubel, supra, chapters 10, 12, 13, 16, 18, and 20.

In preferred embodiments: the antibody is purified: (1) to greater than 95% by weight of the antibody, and in a further preferred embodiment, greater than 99% by weight, as determined, for example, by the Lowry method, UV-Vis spectroscopy, or by SDS-capillary gel electrophoresis (e.g., on a Caliper LabChip GXII, GX 90, or Biorad Bioanalyzer apparatus), (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Isolated naturally occurring antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody can be prepared by at least one purification step.

Treatment

The method of treatment involves administering to a subject in need of treatment a therapeutically effective amount of an antibody or antigen-binding fragment thereof or variant thereof contemplated by the present invention. A "therapeutically effective" amount is defined herein as an amount of the antibody or antigen-binding fragment, alone or in combination with other agents, sufficient to reduce the proliferation of CEACAM6-positive cells or the size of a tumor expressing CEACAM6 in the treated area of the subject, resulting in amelioration of the adverse condition, but in an amount that is toxicologically tolerable. The subject can be a human or non-human animal (e.g., rabbit, rat, mouse, dog, monkey or other lower primate).

One embodiment of the present invention is to provide an antibody or antigen-binding fragment thereof for use as a medicament.

One embodiment of the present invention is to provide an antibody or antigen-binding fragment thereof for use as a medicament for treating cancer. In a preferred embodiment, the cancer is a tumor, and in a highly preferred embodiment, the cancer is a solid tumor.

One embodiment of the present invention is the use of the antibody or antigen-binding fragment thereof in the preparation of a medicament for treating a disease.

One embodiment of the present invention is the use of the antibody or antigen-binding fragment thereof in the preparation of a medicament for treating cancer. In a preferred embodiment, the cancer is a tumor, and in a highly preferred embodiment, the cancer is a solid tumor.

The antibodies or antigen-binding fragments thereof of the present invention can be used as therapeutic or diagnostic tools in various conditions with aberrant CEACAM6 signaling, for example, cell proliferative disorders such as cancer or fibrotic diseases. Disorders and conditions particularly suitable for treatment with the antibodies of the present invention are solid tumors, such as breast cancer, respiratory tract cancer, brain cancer, reproductive organ cancer, digestive tract cancer, urinary tract cancer, eye cancer, liver cancer, skin cancer, head and neck cancer, thyroid cancer, parathyroid cancer, and their distant metastases. Those conditions also include lymphomas, sarcomas, and leukemias.

Digestive tract cancers include, but are not limited to, anal cancer, colon cancer, colorectal cancer, esophageal cancer, gallbladder cancer, stomach cancer, pancreatic cancer, rectal cancer, small intestine cancer, and salivary gland cancer.

Examples of esophageal cancer include, but are not limited to, esophageal cell carcinoma and adenocarcinoma, as well as squamous cell carcinoma, leiomyosarcoma, malignant melanoma, rhabdomyosarcoma, and lymphoma.

Examples of gastric cancer include, but are not limited to, intestinal-type and diffuse-type gastric adenocarcinoma.

Examples of pancreatic cancer include, but are not limited to, ductal adenocarcinoma, adenosquamous carcinoma, and pancreatic endocrine tumors.

Examples of breast cancer include, but are not limited to, triple-negative breast cancer, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ.

Examples of respiratory tract cancers include, but are not limited to, small cell lung cancer and non-small cell lung cancer, as well as bronchial adenoma and pleuropulmonary blastoma.

Examples of brain cancers include, but are not limited to, brainstem and hypothalamic gliomas, cerebellar and cerebral astrocytomas, glioblastomas, medulloblastomas, ependymomas, as well as neuroectodermal and pineal tumors.

Male reproductive organ tumors include, but are not limited to, prostate cancer and testicular cancer. Female reproductive organ tumors include, but are not limited to, endometrial cancer, cervical cancer, ovarian cancer, vaginal cancer, vulvar cancer, and uterine sarcoma.

Examples of ovarian cancer include, but are not limited to, serous tumors, endometrial tumors, mucinous cystadenocarcinomas, granulosa cell tumors, Sertoli-Leydig cell tumors, and androblastoma.

Examples of cervical cancer include, but are not limited to, squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, small cell carcinoma, neuroendocrine tumors, glassy cell carcinoma, and tubulovillous adenocarcinoma.

Tumors of the urinary tract include, but are not limited to, bladder cancer, penile cancer, kidney cancer, renal pelvis cancer, ureter cancer, urethral cancer, and hereditary and sporadic papillary renal cancer.

Examples of renal cancer include, but are not limited to, renal cell carcinoma, urothelial carcinoma, juxtaglomerular cell tumor (reninoma), angiomyolipoma, renal oncocytoma, Bellini duct carcinoma, clear cell sarcoma of the kidney, mesoblastic nephroma, and Wilms' tumor.

Examples of bladder cancer include, but are not limited to, transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, sarcoma, and small cell carcinoma.

Eye cancers include, but are not limited to, intraocular melanoma and retinoblastoma.

Examples of liver cancer include, but are not limited to, hepatocellular carcinoma (liver cell carcinoma with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixed hepatocellular cholangiocarcinoma.

Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer.

Head and neck cancers include, but are not limited to, squamous cell carcinoma of the head and neck, laryngeal cancer, hypopharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, salivary gland cancer, lip cancer, and oral cavity cancer, and squamous cell carcinoma.

Lymphomas include, but are not limited to, AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Burkitt lymphoma, Hodgkin's disease, and central nervous system lymphoma.

Sarcomas include, but are not limited to, soft tissue sarcomas, osteosarcomas, malignant fibrous histiocytomas, lymphosarcoma, and rhabdomyosarcoma.

Leukemias include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia.

In a preferred embodiment, the antibodies or antigen-binding fragments thereof of the present invention are suitable for a therapeutic or diagnostic method for the treatment or diagnosis of a cancer disease selected from the group consisting of colorectal cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), pancreatic cancer, gastric cancer, breast cancer and multiple myeloma.

In addition, the antibodies or antigen-binding fragments thereof of the present invention can also be used as therapeutic or diagnostic tools in a variety of other conditions in which CEACAM6 is involved, such as, but not limited to, lung infections, e.g., influenza, Crohn's disease, inflammatory bowel disease, psoriasis, cystic fibrosis, prevention of bacterial entry into the gastrointestinal tract, trauma, hemorrhagic burns, surgery, stroke, myocardial infarction, sepsis, pneumonia, vaccination against infection and cancer, and chronic viral infections.

The above-mentioned disorders are well characterized in humans, but also exist with similar etiology in other animals, including mammals, and can be treated by administering the pharmaceutical compositions of the present invention.

The antibodies or antigen-binding fragments thereof or variants thereof of the present invention can be co-administered with known drugs, and in some cases, the antibodies themselves can be modified. For example, the antibodies or antigen-binding fragments thereof or variants thereof can be conjugated with cytotoxic agents or radioisotopes to potentially further increase efficacy.

The antibodies, or antigen-binding fragments thereof, or variants thereof of the present invention can be administered as a single agent or in combination with one or more additional therapeutic agents, wherein the combination does not cause unacceptable adverse reactions. The combination therapy includes administering a single pharmaceutical dosage form containing the antibodies, or antigen-binding fragments thereof, or variants thereof of the present invention and one or more additional therapeutic agents, as well as administering the antibodies of the present invention and each additional therapeutic agent in its own separate pharmaceutical dosage form. For example, the antibodies, or antigen-binding fragments thereof, or variants thereof of the present invention and the therapeutic agent can be administered to the patient together in a single liquid composition, or each agent can be administered in a separate dosage form.

When separate dosage forms are used, the antibody or antigen-binding fragment thereof or variant thereof of the invention and the one or more additional therapeutic agents can be administered at essentially the same time (eg, simultaneously) or at separately staggered times (eg, sequentially).

Specifically, the antibodies or antigen-binding fragments thereof or variants thereof of the present invention can be used in fixed combination or separate combination with other anti-tumor agents, such as alkylating agents, antimetabolites, plant-derived anti-tumor agents, hormone therapy agents, topoisomerase inhibitors, immunotherapies, antibodies, antibody drugs, biological response modifiers, anti-angiogenic compounds, cell therapy and other anti-tumor drugs, including but not limited to camptothecin derivatives, kinase inhibitors, targeted drugs.

In this regard, the following is a non-limiting list of examples of second agents that can be used in combination with the antibodies of the invention:

Alkylating agents include, but are not limited to, nitrogen mustard N-oxide, cyclophosphamide, ifosfamide, thiotepa, ranimustine, nimustine, temozolomide, altretamine, apaziquone, brostallicin, bendamustine, carmustine, estramustine, fotemustine, glufosfamide, mafosfamide, bendamustin, and mitolactol; platinum-coordinated alkylating compounds include, but are not limited to, cisplatin, carboplatin, epazote, lobaplatin, nedaplatin, oxaliplatin, and satraplatin;

Antimetabolites include, but are not limited to, methotrexate, 6-mercaptopurine nucleoside, mercaptopurine, 5-fluorouracil (alone or in combination with folinic acid), tegafur, doxifluridine, carmofur, cytarabine, cytarabine ocfosfate, enocitabine, gemcitabine, fludarabine, 5-azacitidine, capecitabine, cladribine, clofarabine, decitabine, eflornithine, ethynylcytidine, cytosine arabinoside, cytosine octano ... arabinoside, hydroxyurea, melphalan, nelarabine, nolatrexed, ocfosfite, pemetrexed disodium, pentostatin, pilitrexol, raltitrexed, triapine, trimetrexate, vidarabine, vincristine, and vinorelbine;

Hormonal therapy agents include, but are not limited to, exemestane, Lupron, anastrozole, doxercalciferol, fadrozole, formestane, 11-beta hydroxysteroid dehydrogenase 1 inhibitors, 17-alpha hydroxylase/17,20 lyase inhibitors such as abiraterone acetate, 5-alpha reductase inhibitors such as finasteride and epristeride, antiestrogens such as tamoxifen citrate, and steroids. citrate and fulvestrant, Trelstar, toremifene, raloxifene, lasofoxifene, letrozole, antiandrogens such as bicalutamide, flutamide, mifepristone, nilutamide, Casodex, and antiprogestins, and combinations thereof;

Antitumor substances of plant origin include, for example, those selected from mitotic inhibitors, for example, epothilones such as sagopilone, ixabepilone and epothilone B, vinblastine, vinflunine, docetaxel and paclitaxel;

Cytotoxic topoisomerase inhibitors include, but are not limited to, aclarubicin, doxorubicin, amonafide, belotecan, camptothecin, 10-hydroxycamptothecin, 9-aminocamptothecin, diflomotecan, irinotecan, topotecan, edotecarin, epimbicin, etoposide, exatecan, gimatecan, lurtotecan, mitoxantrone, pirambicin, pixantrone, rubitecan, sobuzoxane, tafluposide, and combinations thereof;

Immunological drugs include interferons (such as interferon α, interferon α-2a, interferon α-2b, interferon β, interferon γ-1a and interferon γ-n1), GM-CSF and other immunopotentiators such as L19-IL2 and other IL2 derivatives, filgrastim, lentinan, sizofilan, TheraCys, ubenimex, aldesleukin, alemtuzumab, BAM-002, dacarbazine, daclizumab, denileukin, gemtuzumab, ozogamicin, ibritumomab, imiquimod, and tebuconazole. miquimod), lenograstim, lentinan, Corixa, molgramostim, sargramostim, tasonermin, tecleukin, thymalasin, tositumomab, Vimlizin, epratuzumab, mitumomab, oregovomab, pemtumomab, and Provenge; ALNX6000, Urelumab, PF-005082566, Galunisertib, AZ10606120, NF340, and BMS-777607;

Immunological drugs also include drugs targeting immune checkpoint regulators or co-inhibitory receptors, including but not limited to CTLA-4, PD1, PD-L1, B7-H3 receptor, B7-H4 receptor, BTLA, TIM3, LAG3, KIRDL, 2B4, VISTA, CD244, CD160, TIGIT, CEACAM1, CEACAM5, and HHLA2. Specifically, some of these drugs are Ipilimumab, Tremelimumab, Nivolumab, Pembrolizumab, Pidilizumab, AMP-224, AMP-514, PDR001, MDX1105, BMS-936,559, Atezolizumab, Medi4736, Avelumab, MSB0010718C, MGA271, IMP321, BMS-986,016, Baviximab, MNRP1685A, Celecoxib, PF-04418948, RQ-15986;

Immunological drugs also include activators of costimulatory receptors, including but not limited to the following: CD28, ICOS, 4-1BB, OX40, CD27, KIRDS, GITR, HVEM, TNFRSF25, CD40L, TMIGD2, TIM-1, CEACAM1, CEACAM5. Among those drugs are CP-870893, Lucatumumab, Dacetuzumab, anti-OX40, MEDI0562, MEDI6469, MEDI6383, CDX-1127, TRX518, Varlilumab;

Immunological drugs also include agents that regulate Treg activity, including but not limited to the following: FOXP3, CD25, and CCR4. These drugs include daclizumab;

Immunological drugs also include agents that regulate the activity of myeloid-derived suppressor cells, including but not limited to drugs targeting CSF1R. An example is emactuzumab, Taladafil;

Immunological drugs also include agents that regulate innate immune cell responses, including agents targeting Toll-like receptors, including but not limited to TLR3, TLR4, TLR7, TLR8, TLR9, NGK2A, and NKG2D. These drugs include, for example, imiquimod, CPG7909 (PF-3512676, CPG2006); MGN1703, SD-101, hiltonol (polyICLC), anti-NGK2A (IPH2201), OM-174, 852A, VTX-2337, and IMO-2055.

Immunological drugs also include agents that regulate innate immune cell responses, including drugs targeting the following, but not limited to: CSF-1, CSF1R, KIR, ILTs, LIRs, MICA, MICB, CD244, CCL2, CD47. Specifically, some of these drugs are anti-KIR (IHP2101; IPH2101; 1-7F9); Lirilumab (IPH2012; BMS-986,015); Carlumab (CNTO888), IMC-CS4, FPA008, PLX3397, ARRY-382, CC-90002, anti-CD47 (Hu5F9-G4), BLZ945;

Immunological drugs also include agents for retargeting immune cells, including but not limited to bispecific antibodies, Darts, such as those targeting B7-H3, Bites, such as CD19xCD3; such as Removab anti-EPCAMxCD3xFC, NK cell targeting agents;

Immunotherapy also includes agents that modulate the tumor microenvironment and improve immune cell infiltration and response, including vaccines and adjuvants, and is not limited to GVAX and FVAX;

In this regard, vaccines include dendritic cell-based vaccines, viral vaccines, mRNA-based vaccines, and peptide-based vaccines;

Immunological drugs also include agents for improving immune cell infiltration, including but not limited to IFN-g, IL15, IL21, IL2, CXCR4, CXCl12. Some of these drugs are Denenicokin (BMS982, 470), ALT-803, hetIL15, Ulocuplumab, BKT140, CXCR2-specific mab, AD3100, Maravirox, PF-4136309;

Immunological drugs also include agents or methods that improve the priming and activation of APCs and T cells, including drugs targeting, but not limited to, m-TOR GSK3β inhibitors, loaded DCs (e.g., Provenge), radiotherapy, external irradiation;

Immunological drugs also include kynurenine pathway modulators, including drugs targeting the following, but not limited to: IDO1, IDO2, TDO. Among those drugs are INCB024360, Indoximod (NLG8189; 1-methyl-D-tryptophan, D-1MT), GDC-0919, NLG919, LM10;

Immunological drugs also include adenosine pathway modulators, including but not limited to the following: CD39, CD73, A2A receptor, A2B receptor. Such drugs include compound 9, NCX-4016, AT38, SCH58261, SCH420814, PSB1115, ARL67176, AMPCP;

Immunotherapy also includes TGFβ/ALK5 pathway regulators. Such drugs include OY2157299 and EW-7187;

Immune drugs also include Sting activators;

Biological response modifiers are agents that modify the defense mechanisms or biological responses of living organisms (such as the survival, growth, or differentiation of tissue cells) to direct them to have anti-tumor activity; such agents include, for example, krestin, lentinan, sizoran, picibanil, ProMune, and ubenimex;

Anti-angiogenic compounds include, but are not limited to, acitretin, aflibercept, angiostatin, aplidine, asentar, axitinib, bevacizumab, brivanib, alaninat, cilengtide, combretastatin, endostatin, fenretinide, halofuginone, pazopanib, ranibizumab, rebimastat, recentin, regorafenib, removab, revlimid, sorafenib, squalamine, sunitinib, telatinib, thalidomide, ukrain, vatalanib, and vitaxin;

Anti-angiogenic agents also include VEGF inhibitors, including but not limited to sorafenib, regorafenib, bevacizumab, sunitinib, recentin, axitinib, aflibercept, telatinib, brivanib alanine (brivanibalaninate), vatalanib, pazopanib and ranibizumab;

Antibody drugs include, but are not limited to, trastuzumab, cetuximab, bevacizumab, rituximab, ticilimumab, ipilimumab, tremelimumab, pembrolizumab, nivolumab, pidilizumab, RG-7446/MPDL3280A, BMS-936559 (MDX1105), durvalumab (Medi-4736), MSB-0010718C, ruxolitinib, catumaxomab, atacicept, ogavuzumab, panitumumab, and alemtuzumab;

Antibody drugs also include antibody drug conjugates, including but not limited to those targeting: mesothelin, C4.4A, FGFR2, HER2, PSMA;

Antibody drugs also include thorium-targeted conjugates, including but not limited to those targeting: mesothelin, C4.4A, FGFR2, HER2, PSMA, CEACAM6;

Antibody drugs also include bispecific (or multispecific) antibody formats, including but not limited to bispecific (or multispecific) IgG and bispecific (or multispecific) antibody fragments and protein fusions and conjugates thereof (e.g., CrossMab, DAF (2 in 1), DAF (4 in 1), DutaMab, DT-IgG, KiH assembled IgG, charge pair assembled IgG, KiH-common LC, Fab-arm exchange, SEED body, Triomab, LUZ-Y, Fcab, κλ-mAb, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH-IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zy body, DVI-IgG (4-in-1), dinanobody, BiTE, diabody, DART, DART-Fc, TandAb, scdiabody, scdiabody-CH3, diabody-CH3, triabody, minibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scdiabody-Fc, diabody-Fc, tandem scFv-Fc, intrabody, dock and lock fusion, ImmTAC, HASbody, scdiabody-HAS, tandem scFv-toxin, IgG-IgG, Cov-X-body, scFv1-PEG-scFv2 and others);

Antibody drugs also include recombinant proteins with antibody-like binding properties produced by recombinant technology, such as but not limited to DARPIN molecules;

Cellular therapies include, but are not limited to, tumor-infiltrating lymphocytes isolated from cancer patients, such as ex vivo stimulated T cells, e.g., Sipuleucel-T;

Cellular therapies include, but are not limited to, tumor-infiltrating lymphocytes isolated from cancer patients such as Sipuleucel-T and genetically engineered T cells carrying chimeric antigen receptors (CARs), such as, for example, CD19-CAR-T cells; CAR-Her2-T-cells;

Other anticancer agents include, for example, alitretinoin, ampligen, atrasentan, bexarotene, bortezomib, bosentan, calcitriol, exisulind, fotemustine, ibandronic acid, miltefosine, mitoxantrone, I-asparaginase, procarbazine, dacarbazine, hydroxyurea, pegaspargase, pentostatin, tazaroten, velcade, gallium nitrate, canfosfamide, darinaparsin, and tretinoin, PI3065, TG100-115;

EGFR (HER1) inhibitors such as, for example, cetuximab, panitumumab, vectibix, gefitinib, erlotinib, and Zactima;

HER2 inhibitors such as, for example, lapatinib, tratuzumab, and pertuzumab;

mTOR inhibitors such as, for example, temsirolimus, sirolimus/rapamycin, and everolimus;

c-Met inhibitors;

PI3K inhibitors such as PI3K inhibitor 1 (2-amino-N-[7-methoxy-8-(3-morpholin-4-ylpropoxy)-2,3-dihydroimidazo[1,2-c]quinazolin-5-yl]pyrimidine-5-carboxamide dihydrochloride (see compounds of Examples 1 and 2 of WO 2012/136553)

and AKT inhibitors;

CDK inhibitors such as roscovitine and flavopiridol;

Spindle assembly checkpoint inhibitors and targeted antimitotic agents such as PLK inhibitors, Aurora inhibitors (e.g., Hesperadin), checkpoint kinase inhibitors, and KSP inhibitors;

BRAFV600E inhibitors such as vemurafenib and dabrafenib;

HDAC inhibitors such as, for example, panobinostat, vorinostat, MS275, belinostat, and LBH589;

HSP90 and HSP70 inhibitors;

Proteasome inhibitors such as bortezomib and carfilzomib;

Serine/threonine kinase inhibitors include MEK inhibitors and Raf inhibitors such as sorafenib;

Farnesyl transferase inhibitors such as, for example, tipifarnib;

Tyrosine kinase inhibitors include, for example, dasatinib, nilotibib, regorafenib, bosutinib, sorafenib, bevacizumab, sunitinib, cediranib, axitinib, aflibercept, telatinib, imatinib mesylate, brivanib alanine, pazopanib, ranibizumab, vatalanib, cetuximab, panitumumab, vectibix, gefitinib, erlotinib, lapatinib, trastuzumab, pertuzumab, and c-Kit inhibitors;

Vitamin D receptor agonists;

Bcl-2 protein inhibitors such as obatoclax, oblimersen sodium, and gossypol;

Cluster of differentiation 20 receptor antagonists such as, for example, rituximab;

Ribonucleotide reductase inhibitors such as, for example, gemcitabine;

Tumor necrosis factor-related apoptosis-inducing ligand receptor 1 agonists such as, for example, mapatumumab;

Tumor necrosis factor-related apoptosis-inducing ligand receptor 2 agonists such as, for example, lexatumumab, conatumumab, CS-1008, PRO95780;

Serotonin receptor antagonists such as, for example, rEV598, xaliprode, palonosetron hydrochloride, granisetron, Zindol, and AB-1001;

Integrin inhibitors include α5-β1 integrin inhibitors such as, for example, E7820, JSM 6425, volociximab, and endostatin;

androgen receptor antagonists include, for example, nandrolone decanoate, fluoxymesterone, methyltestosterone (Android), Prost-aid, andromustine, bicalutamide, flutamide, apo-cyproterone, apo-flutamide, chlormadinone acetate, Androcur, Tabi, cyproterone acetate, and nilutamide;

aromatase inhibitors such as, for example, anastrozole, letrozole, testolactone, exemestane, aminoglutethimide, and formestane;

Matrix metalloproteinase inhibitors;

Furthermore, the antibodies of the present invention can be combined with means that cause immunogenic cell death, including but not limited to ultraviolet light, oxidative treatment, heat shock, targeted and non-targeted radiation therapy, shikonin, high hydrostatic pressure, oncolytic viruses, and photodynamic therapy;

Furthermore, the antibodies of the invention can be combined with agents that cause immunogenic cell death, including but not limited to sunitinib, JAK2 inhibitors, anthracyclines, doxorubicin, mitoxantrone, oxaliplatin and cyclophosphamide, targeted and non-targeted microtubule destabilizing drugs (such as, for example, auristatins and maytansinoids);

The compounds of the present invention may also be used in combination with radiotherapy and/or surgical intervention for cancer treatment;

Furthermore, the antibodies of the present invention may be used as such or in compositions, research and diagnostics, or as analytical reference standards, etc., which are well known in the art.

Diagnostic methods

Anti-CEACAM6 antibodies or antigen-binding fragments thereof can be used to detect the presence of tumors expressing CEACAM6. The presence of CEACAM6-containing cells or shed CEACAM6 in various biological samples (including serum and tissue biopsy samples) can be detected using anti-CEACAM6 antibodies. In addition, anti-CEACAM6 antibodies can be used in various imaging methods, such as immunoscintigraphy using antibodies conjugated with ^99Tc (or other isotopes). For example, imaging protocols similar to those described using ^111In -conjugated anti-PSMA antibodies can be used to detect pancreatic or ovarian cancer (Sodee et al., Clin. Nuc. Med. 21: 759-766, 1997). Another detection method that can be used is positron emission tomography (see Herzog et al., J. Nucl. Med. 34: 2222-2226, 1993) by conjugating the antibodies of the invention to a suitable isotope.

Pharmaceutical compositions and administration

In order to treat any of the aforementioned diseases, one or more physiologically acceptable carriers or excipients can be used to prepare the pharmaceutical composition used according to the present invention in a conventional manner. According to the disease type treated, the antibody of the present invention or its antigen-binding fragment can be used by any suitable means (it can change). Possible routes of administration include parenteral (for example, intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous), intrapulmonary and intranasal, and if necessary, for local immunosuppressive therapy, intralesional administration (intralesional administration). In addition, the antibody of the present invention or its antigen-binding fragment or its variant can be used by pulse infusion with the antibody of for example decrement. Preferably, dosage is given by injection (most preferably intravenous or subcutaneous injection), and this part depends on whether it is short-term administration or long-term administration. The amount to be administered will depend on multiple factors, such as clinical symptoms, individual body weight, whether to use other drugs. The technician will recognize that route of administration can change according to disease to be treated or the patient's condition.

One embodiment of the present invention is a pharmaceutical composition comprising an anti-CEACAM6 antibody, or antigen-binding fragment thereof, or variant thereof, alone or in combination with at least one other agent (such as a stabilizing compound), which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. A further embodiment is a pharmaceutical composition comprising a CEACAM6-binding antibody, or antigen-binding fragment thereof, and other pharmaceutically active compounds suitable for treating CEACAM6-related diseases (such as cancer). Any of these molecules can be administered to a patient alone or in combination with other agents, drugs, or hormones, in a pharmaceutical composition in which they are mixed with one or more excipients or pharmaceutically acceptable carriers. In one embodiment of the present invention, the pharmaceutically acceptable carrier is pharmaceutically inert.

The present invention further relates to the use of pharmaceutical compositions. Such use is achieved orally or parenterally. The method of parenteral delivery includes local, intra-arterial (directly to the tumor), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal or intranasal administration. In addition to the active ingredient, these pharmaceutical compositions can also contain suitable pharmaceutically acceptable carriers (including excipients and adjuvants), which are conducive to processing the active compound into pharmaceutically usable preparations. For further details on the technology used to prepare and use, see the latest version of Remington's Pharmaceutical Sciences (editor Maack Publishing Co, Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulated in dosages suitable for oral administration using pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, lozenges, capsules, liquids, gels, syrups, slurries, suspensions, etc., which are ingested by the patient.

Pharmaceutical preparations for oral use can be obtained by mixing the active compound with a solid excipient, optionally grinding the resulting mixture, and processing the mixture of particles, after adding suitable adjuvants (if necessary), to obtain tablets or lozenge cores. Suitable excipients are carbohydrates or protein fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; starch from corn, wheat, rice, potato or other plants; cellulose, such as methylcellulose, hydroxypropyl methylcellulose or sodium carboxymethylcellulose; and gums including gum arabic and tragacanth; and proteins such as gelatin and collagen. If necessary, disintegrants or solubilizers such as cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereof, such as sodium alginate, can be added.

Dragee cores may be provided with a suitable coating, such as a concentrated sugar solution, which may also contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol and/or titanium dioxide, lacquer solution and a suitable organic solvent or solvent mixture. Dyes or pigments may be added to the tablet or dragee coating for product identification or to characterize the amount of active compound, i.e., the dosage.

Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin and sealed soft capsules made of gelatin and a coating such as glycerol or sorbitol. Push-fit capsules can contain the active ingredient mixed with a filler or binder such as lactose or starch, a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the active compound can be dissolved or suspended in a suitable liquid such as a fatty oil, liquid paraffin, or liquid polyethylene glycol, with or without a stabilizer.

Pharmaceutical preparations for parenteral administration include aqueous solutions of the active compound. For injection, the pharmaceutical composition of the present invention can be prepared in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution or physiologically buffered saline. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. In addition, the suspension of the active compound can be prepared as a suitable oily injection suspension. Suitable lipophilic solvents or vehicles include fatty oils (such as sesame oil) or synthetic fatty acid esters (such as ethyl oleate or triglycerides) or liposomes. Optionally, the suspension can also contain a suitable stabilizer or increase the solubility of the compound to allow the preparation of a highly concentrated solution.

For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention may be manufactured in a manner known in the art, eg, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

The pharmaceutical composition can be provided as a salt and can be formed together with an acid, including but not limited to hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, etc. Salts tend to be more soluble in aqueous solvents or other protic solvents (which are the corresponding free base forms). In other cases, a preferred formulation can be a lyophilized powder in 1 mM-50 mM histidine or phosphate or Tris, 0.1%-2% sucrose and/or 2%-7% mannitol in a pH range of 4.5-7.5, optionally containing additional substances such as polysorbate, which is mixed with a buffer before use.

After a pharmaceutical composition comprising a compound of the invention formulated in an acceptable carrier has been prepared, it can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of an anti-CEACAM6 antibody or antigen-binding fragment thereof, this labeling can include the amount, frequency, and method of administration.

medicine box

The present invention further relates to pharmaceutical packs and kits comprising one or more containers containing one or more of the ingredients of the compositions of the present invention as described above. The one or more containers may be accompanied by a notice in a format prescribed by a governmental agency regulating the manufacture, use, or sale of drugs or biological products, reflecting approval by the regulatory agency for manufacture, use, or sale of the product for human administration.

Therapeutically effective dose

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose, ie, treatment of a particular disease state characterized by CEACAM6 expression.

Determination of an effective dosage is well within the capability of those skilled in the art.

Determine the therapeutically effective amount of the novel antibody of the present invention or its antigen-binding fragment or its variant, greatly depending on the nature of the specific patient characteristics, route of administration and the disease to be treated. General guidance can be found in, for example, the International Conference on Harmonization publication and Remington's Pharmaceutical Sciences, Chapter 27 and 28, pages 484-528 (18th edition, Alfonso R. Gennaro, Ed., Easton, Pa.: Mack Pub. Co., 1990). More specifically, the determination of the therapeutically effective amount depends on factors such as the toxicity and efficacy of the drug. Toxicity can be measured using methods well known in the art, and can be found in the aforementioned references. The same guidance can be used to measure efficacy in conjunction with the method described in the examples below.

For any compound, the therapeutically effective dose can be estimated initially in cell culture assays (e.g., neoplastic cells) or in animal models (usually mice, rabbits, dogs, pigs, or monkeys). Animal models are also used to achieve the desired concentration range and route of administration. This information can then be used to determine useful doses and routes for administration in humans.

A therapeutically effective dose refers to the amount of an antibody or antigen-binding fragment thereof that improves a symptom or condition. The therapeutic efficacy and toxicity of such compounds can be determined in cell cultures or experimental animals by standard pharmaceutical procedures, such as _ED50 (the dose that is therapeutically effective in 50% of the population) and _LD50 (the lethal dose for 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the _ED50 / _LD50 ratio. Pharmaceutical compositions that exhibit a large therapeutic index are preferred. Data obtained from cell culture assays and animal studies are used to determine a dosage range for human use. The dosage of such compounds is preferably within a circulating concentration range that is substantially nontoxic or nontoxic, including _ED50 . Depending on the dosage form employed, the sensitivity of the patient, and the route of administration, the dosage varies within this range.

The exact dosage is chosen by each physician, taking into account the patient to be treated. Dosage and administration are adjusted to provide adequate levels of the active moiety or to maintain the desired effect. Additional factors that may be considered include the severity of the disease state, such as tumor size and location; the patient's age, weight, and sex; diet, time and frequency of administration, drug combination, reaction sensitivity, and tolerance/response to treatment. Long-acting pharmaceutical compositions may be administered, for example, every 3 to 4 days, weekly, once every two weeks, or once every three weeks, depending on the half-life and clearance rate of the particular formulation.

Depending on the route of administration, normal doses may vary from 0.1 micrograms to 100,000 micrograms, up to a total dose of about 10 g. Guidance on specific doses and delivery methods is provided in the literature. See U.S. Patent Nos. 4,657,760; 5,206,344; or 5,225,212. One skilled in the art may employ polynucleotide formulations other than proteins or their inhibitors. Similarly, the delivery of polynucleotides or polypeptides is specific for specific cells, conditions, sites, and the like. The preferred specific activity range of radiolabeled antibodies may be 0.1-10 mCi/mg protein (Riva et al., Clin. Cancer Res. 5:3275-3280, 1999; Ulaner et al., 2008 Radiology 246(3):895-902).

A further preferred embodiment of the present invention is:

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to human CEACAM6 and cynomolgus monkey CEACAM6.

2. An isolated antibody or antigen-binding fragment thereof that specifically binds to the mature extracellular domain of human CEACAM6 (represented by amino acids at positions 35-320 of SEQ-ID No: 179) and the mature extracellular domain of cynomolgus monkey CEACAM6 (represented by amino acids at positions 35-320 of SEQ-ID No: 177).

3. An isolated antibody or antigen-binding fragment thereof that specifically binds to human CEACAM6 domain 1 (represented by amino acids 35-142 of SEQ-ID No: 179) and cynomolgus monkey CEACAM6 domain 1 (represented by amino acids 35-142 of SEQ-ID No: 177).

4. An isolated antibody or antigen-binding fragment thereof that specifically binds to a protein comprising human CEACAM6 domain 1 (represented by amino acids 35-142 of SEQ-ID No: 179) and a protein comprising cynomolgus monkey CEACAM6 domain 1 (represented by amino acids 35-142 of SEQ-ID No: 177).

5. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4, which does not significantly cross-react with human CEACAM1, human CEACAM3 and human CEACAM5.

6. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 5, which is capable of binding to human and cynomolgus monkey CEACAM6 or its mature extracellular domain or domain 1 thereof with an affinity that is within one order of magnitude of the monovalent _KD .

7. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, which competes for binding with the 9A6 antibody (Genovac/Aldevron) on human CEACAM6.

8. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, which interferes with the interaction between CEACAM6 and CEACAM1.

9. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, which is capable of altering the cytokine profile of tumor antigen-specific T cells towards a more activated phenotype, wherein the more activated phenotype is characterized by an increase in IFN-γ secretion compared to a control sample, preferably an increase of ≥1.5 times (1.5 times or more).

10. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, which is capable of inducing immune modulation.

11. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, which is capable of alleviating CEACAM6-mediated immunosuppression of tumor antigen-specific T cells, as measured by IFN-γ secretion of tumor antigen-specific T cells or the number of IFN-γ-secreting activated T cells.

12. An antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, which is capable of altering the cytokine profile of tumor antigen-specific T cells towards a more cytotoxic and/or activated phenotype, wherein the more cytotoxic and/or activated phenotype is characterized by increased secretion of IFN-γ and/or IL-2 and/or TNF-α, preferably an increase in secretion of IFN-γ and/or IL-2 and/or TNF-α by ≥1.5 times (1.5 times or more) compared to the control sample.

13. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, which binds to an epitope of human CEACAM6, wherein the epitope comprises one or more amino acid residues selected from Gln60, Asn61, Arg62, Ile63, Val83, Ile84, Gly85, Thr90, Ser127, Asp128 and Leu129 of SEQ ID NO: 179.

14. The antibody or antigen-binding fragment thereof according to embodiment 13, which binds to an epitope of human CEACAM6, wherein the epitope comprises amino acid residues Gln60, Asn61, Arg62, Ile63, Val83, Ile84, Gly85, Thr90, Ser127, Asp128 and Leu129 of SEQ ID NO: 179.

15. The antibody or antigen-binding fragment thereof according to embodiment 13 or 14, which binds to a human CEACAM6 protein comprising an Ile63Leu mutation and does not bind to a human CEACAM6 protein comprising an Ile63Phe mutation numbered according to SEQ ID NO: 179.

16. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, comprising:

i. a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 48, H-CDR2 comprising SEQ ID NO: 49, and H-CDR3 comprising SEQ ID NO: 50, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 52, L-CDR2 comprising SEQ ID NO: 53, and L-CDR3 comprising SEQ ID NO: 54, or

ii. a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 106, H-CDR2 comprising SEQ ID NO: 107, and H-CDR3 comprising SEQ ID NO: 108, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 110, L-CDR2 comprising SEQ ID NO: 111, and L-CDR3 comprising SEQ ID NO: 112, or

iii. a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 4, H-CDR2 comprising SEQ ID NO: 5, and H-CDR3 comprising SEQ ID NO: 6, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 8, L-CDR2 comprising SEQ ID NO: 9, and L-CDR3 comprising SEQ ID NO: 10, or

iv. a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 34, H-CDR2 comprising SEQ ID NO: 35, and H-CDR3 comprising SEQ ID NO: 36, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 38, L-CDR2 comprising SEQ ID NO: 39, and L-CDR3 comprising SEQ ID NO: 40, or

v. a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 120, H-CDR2 comprising SEQ ID NO: 121, and H-CDR3 comprising SEQ ID NO: 122, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 124, L-CDR2 comprising SEQ ID NO: 125, and L-CDR3 comprising SEQ ID NO: 126, or

vi. a heavy chain antigen-binding region comprising an H-CDR1 comprising SEQ ID NO: 24, an H-CDR2 comprising SEQ ID NO: 25, and an H-CDR3 comprising SEQ ID NO: 26, and a light chain antigen-binding region comprising an L-CDR1 comprising SEQ ID NO: 28, an L-CDR2 comprising SEQ ID NO: 29, and an L-CDR3 comprising SEQ ID NO: 30, or

vii. a heavy chain antigen-binding region comprising an H-CDR1 comprising SEQ ID NO: 76, an H-CDR2 comprising SEQ ID NO: 77, and an H-CDR3 comprising SEQ ID NO: 78, and a light chain antigen-binding region comprising an L-CDR1 comprising SEQ ID NO: 80, an L-CDR2 comprising SEQ ID NO: 81, and an L-CDR3 comprising SEQ ID NO: 82, or

viii. a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 134, H-CDR2 comprising SEQ ID NO: 135, and H-CDR3 comprising SEQ ID NO: 136, and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 138, L-CDR2 comprising SEQ ID NO: 139, and L-CDR3 comprising SEQ ID NO: 140, or

ix. a heavy chain antigen-binding region comprising an H-CDR1 comprising SEQ ID NO: 148, an H-CDR2 comprising SEQ ID NO: 149, and an H-CDR3 comprising SEQ ID NO: 150, and a light chain antigen-binding region comprising an L-CDR1 comprising SEQ ID NO: 152, an L-CDR2 comprising SEQ ID NO: 153, and an L-CDR3 comprising SEQ ID NO: 154, or

x. a heavy chain antigen-binding region comprising an H-CDR1 comprising SEQ ID NO: 14, an H-CDR2 comprising SEQ ID NO: 15, and an H-CDR3 comprising SEQ ID NO: 16, and a light chain antigen-binding region comprising an L-CDR1 comprising SEQ ID NO: 18, an L-CDR2 comprising SEQ ID NO: 19, and an L-CDR3 comprising SEQ ID NO: 20, or

xi. a heavy chain antigen-binding region comprising an H-CDR1 comprising SEQ ID NO: 62, an H-CDR2 comprising SEQ ID NO: 63, and an H-CDR3 comprising SEQ ID NO: 64, and a light chain antigen-binding region comprising an L-CDR1 comprising SEQ ID NO: 66, an L-CDR2 comprising SEQ ID NO: 67, and an L-CDR3 comprising SEQ ID NO: 68, or

xii. a heavy chain antigen-binding region comprising H-CDR1 comprising SEQ ID NO: 92, H-CDR2 comprising SEQ ID NO: 93, and H-CDR3 comprising SEQ ID NO: 94; and a light chain antigen-binding region comprising L-CDR1 comprising SEQ ID NO: 96, L-CDR2 comprising SEQ ID NO: 97, and L-CDR3 comprising SEQ ID NO: 98.

17. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, comprising:

i. a variable heavy chain sequence as shown in SEQ ID NO: 47 and a variable light chain sequence as shown in SEQ ID NO: 51, or

ii. the variable heavy chain sequence as shown in SEQ ID NO: 105 and the variable light chain sequence as shown in SEQ ID NO: 109, or

iii. a variable heavy chain sequence as shown in SEQ ID NO: 3 and a variable light chain sequence as shown in SEQ ID NO: 7, or

iv. a variable heavy chain sequence as shown in SEQ ID NO: 33 and a variable light chain sequence as shown in SEQ ID NO: 37, or

v. a variable heavy chain sequence as shown in SEQ ID NO: 119 and a variable light chain sequence as shown in SEQ ID NO: 123, or

vi. a variable heavy chain sequence as shown in SEQ ID NO: 23 and a variable light chain sequence as shown in SEQ ID NO: 27, or

vii. a variable heavy chain sequence as shown in SEQ ID NO: 75 and a variable light chain sequence as shown in SEQ ID NO: 79, or

viii. a variable heavy chain sequence as shown in SEQ ID NO: 133 and a variable light chain sequence as shown in SEQ ID NO: 137, or

ix. the variable heavy chain sequence shown in SEQ ID NO: 147 and the variable light chain sequence shown in SEQ ID NO: 151, or

x. the variable heavy chain sequence as shown in SEQ ID NO: 13 and the variable light chain sequence as shown in SEQ ID NO: 17, or

xi. a variable heavy chain sequence as shown in SEQ ID NO: 61 and a variable light chain sequence as shown in SEQ ID NO: 65, or

xii. The variable heavy chain sequence shown in SEQ ID NO: 91 and the variable light chain sequence shown in SEQ ID NO: 95.

18. The antibody according to any of the preceding embodiments, which is an IgG antibody.

19. The antibody of embodiment 18, comprising:

i. a heavy chain region corresponding to SEQ ID NO: 57 and a light chain region corresponding to SEQ ID NO: 58, or

ii. a heavy chain region corresponding to SEQ ID NO: 115 and a light chain region corresponding to SEQ ID NO: 116, or

iii. a heavy chain region corresponding to SEQ ID NO: 43 and a light chain region corresponding to SEQ ID NO: 44, or

iv. a heavy chain region corresponding to SEQ ID NO: 129 and a light chain region corresponding to SEQ ID NO: 130, or

v. a heavy chain region corresponding to SEQ ID NO: 85 and a light chain region corresponding to SEQ ID NO: 86, or

vi. a heavy chain region corresponding to SEQ ID NO: 143 and a light chain region corresponding to SEQ ID NO: 144, or

vii. a heavy chain region corresponding to SEQ ID NO: 157 and a light chain region corresponding to SEQ ID NO: 158, or

viii. a heavy chain region corresponding to SEQ ID NO: 71 and a light chain region corresponding to SEQ ID NO: 72, or

ix. A heavy chain region corresponding to SEQ ID NO: 101 and a light chain region corresponding to SEQ ID NO: 102.

20. The antigen-binding fragment according to embodiments 1 to 17, which is a scFv, Fab, Fab' fragment or F(ab') ₂ fragment.

21. The antibody or antigen-binding fragment according to any one of the preceding embodiments, which is a monoclonal antibody or antigen-binding fragment.

22. The antibody or antigen-binding fragment according to any one of the preceding embodiments, which is a human, humanized or chimeric antibody or antigen-binding fragment.

23. An antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 22.

24. An isolated nucleic acid sequence encoding the antibody or antigen-binding fragment according to any one of embodiments 1 to 22.

25. A vector comprising the nucleic acid sequence according to embodiment 24.

26. An isolated cell expressing the antibody or antigen-binding fragment according to any one of embodiments 1 to 22 and/or comprising the nucleic acid according to embodiment 24 or the vector according to embodiment 25.

27. The isolated cell according to embodiment 26, wherein the cell is a prokaryotic or eukaryotic cell.

28. A method of producing the antibody or antigen-binding fragment according to any one of embodiments 1 to 22, comprising culturing the cell according to embodiment 27 and purifying the antibody or antigen-binding fragment.

29. The antibody or antigen-binding fragment according to any one of embodiments 1 to 22 or the antibody-drug conjugate according to embodiment 23 for use as a medicament.

30. The antibody or antigen-binding fragment according to any one of embodiments 1 to 22 or the antibody-drug conjugate according to embodiment 23 for use as a diagnostic agent.

31. The antibody or antigen-binding fragment according to any one of embodiments 1 to 22 or the antibody-drug conjugate according to embodiment 23 for use as a medicament for the treatment of cancer.

32. The antibody or antigen-binding fragment according to any one of embodiments 1 to 22 or the antibody-drug conjugate according to embodiment 23, for use in the preparation of a medicament for treating a disease.

33. The antibody or antigen-binding fragment according to any one of embodiments 1 to 22 or the antibody-drug conjugate according to embodiment 23, for use in the preparation of a medicament for the treatment of cancer.

34. A pharmaceutical composition comprising the antibody or antigen-binding fragment according to any one of embodiments 1 to 22 or the antibody-drug conjugate according to embodiment 23.

35. A pharmaceutical composition according to embodiment 34 in combination with one or more therapeutically active compounds.

36. A method for treating a disorder or condition associated with the undesired presence of CEACAM6, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition according to embodiment 34 or a combination according to embodiment 35.

37. A method for preparing an anti-CEACAM6 antibody that specifically binds to human CEACAM6 and cynomolgus monkey CEACAM6, the method comprising immunizing an animal, preferably a mouse, with a protein comprising domain 1 of cynomolgus monkey CECAM6 represented by amino acids 35-142 of SEQ-ID NO: 177, determining the amino acid sequence of an antibody that specifically binds to human CEACAM6 and cynomolgus monkey CEACAM6, optionally followed by humanization or generating a chimeric antibody, and recombinantly expressing the antibody.

Example

The present invention is further described by the following examples. These examples are provided for illustration purposes only, with reference to specific embodiments. These examples, while illustrating certain specific aspects of the present invention, are not intended to describe limiting aspects of the disclosed invention or to define the scope of the disclosed invention.

All examples were performed using standard techniques well known and routine to those skilled in the art, unless otherwise described in detail. Conventional molecular biology techniques can be used to perform the following examples, as described in standard laboratory manuals, such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

Example 1: Monkey CEACAM6 sequence and tool generation

A summary of the protein sequences of the antigens and reference compounds used is provided in Table 2:

name Protein-ID describe SEQ-ID Human CEACAM6 TPP-4639 full length SEQ-ID NO:179 Cynomolgus monkey CEACAM6 TPP-4189 full length SEQ-ID NO:177 human CEACAM1 TPP-4185 full length SEQ-ID NO:173 human CEACAM3 TPP-4187 full length SEQ-ID NO:175 human CEACAM5 TPP-4188 full length SEQ-ID NO:176 human CEACAM8 TPP-4190 full length SEQ-ID NO:178 Human CEACAM19 TPP-4186 full length SEQ-ID NO:174 Human CEACAM6 TPP-1436 Extracellular (mature form) (R&D Systems 3934-CM) SEQ-ID NO:162 human CEACAM1 TPP-1437 Extracellular (mature form) (R&D Systems 2244-CM) SEQ-ID NO:163 human CEACAM3 TPP-2755 Extracellular (mature form) (Sino Biological Inc. 11933-H08H) SEQ-ID NO:172 human CEACAM5 TPP-1438 Extracellular (mature form) (R&D Systems 4128-CM) SEQ-ID NO:164 Rhesus monkey CEACAM6-Xa-Fc-His TPP-1306 Extracellular (mature form), fusion with Xa-Fc-His SEQ-ID NO:161 human CEACAM6-Xa-Fc-His TPP-1790 Extracellular (mature form), fusion with Xa-Fc-His SEQ-ID NO:165 humanCEACAM6-Dom1-MacMul-Xa-Fc-His TPP-1791 Extracellular (mature form), fusion with Xa-Fc-His, domain 1 replaced by the corresponding macaque domain SEQ-ID NO:166 HumanCEACAM6-Dom2-MacMul-Xa-Fc-His TPP-1792 Extracellular (mature form), fusion with Xa-Fc-His, domain 2 replaced by the corresponding cynomolgus domain SEQ-ID NO:167 HumanCEACAM6-Dom3-MacMul-Xa-Fc-His TPP-1793 Extracellular (mature form), fusion with Xa-Fc-His, domain 3 replaced by the corresponding macaque domain SEQ-ID NO:168 Human CEACAM6 APP-320 Extracellular (mature form), obtained by cleavage of TPP-1790 with factor Xa Human CEACAM6-Domain 1-His TPP-1794 Domain 1, fusion with His (expressed in E. coli) SEQ-ID NO:169 Cynomolgus monkey CEACAM6-Xa-Fc-His TPP-2443 Extracellular (mature form), fusion with Xa-Fc-His SEQ-ID NO: 170 Cynomolgus monkey CEACAM6 APP-319 Extracellular (mature form), obtained by Factor Xa cleavage of TPP-2443 Cynomolgus monkey CEACAM6-Domain 1-Xa-Fc-His TPP-2452 Domain 1, fusion with Xa-Fc-His SEQ-ID NO:171 Cynomolgus monkey CEACAM6-domain 1 APP-325 Domain 1, obtained by Factor Xa cleavage of TPP-2452 Neo201 (human IgG1) TPP-1173 Based on US20130189268 SEQ-ID NO:1 & SEQ-ID NO:2 Neo201 (human IgG2) TPP-3688 Based on US20130189268 SEQ-ID NO:89 & SEQ-ID:90 9A6 (mouse IgG1) TPP-1744 Based on Genovac/Aldevron (GM-0509) 9A6 (chimeric hIgG1) TPP-1745 Based on Genovac/Aldevron (GM-0509) 9A6 (chimeric hIgG2) TPP-3470 Based on Genovac/Aldevron (GM-0509)

Table 2: Names, protein-IDs, and SEQ-IDs used in this study.

The protein sequences of human CEACAMs were obtained from the UniProtKB/TrEMBL databases: human CEACAM6 (P40199), human CEACAM1 (P13688), human CEACAM3 (P40198), human CEACAM5 (P06731), human CEACAM8 (P31997), and human CEACAM19 (Q7Z692). The macaque (Macaca mulatta) (rhesus monkey) protein sequence for CEACAM6 is also available (F6YVW1). The cynomolgus monkey (Macaca fascicularis) protein sequence for CEACAM6 was deduced from the publicly available nucleotide sequence by: a) applying common intron/exon splicing rules, b) comparing with different monkey/primate protein sequences, and c) considering conservation of genomic structure between humans/primates/monkeys. Cynomolgus monkey CEACAM6 is represented by TPP-4189.

Recombinant extracellular domains of CEACAM were obtained from commercial sources or produced in-house. To this end, the extracellular domains were appended with a Factor Xa cleavage site, a human IgG1 Fc fragment, and a His tag at the C-terminus and expressed in HEK293 cells using standard transient transfection procedures. The proteins were purified from cell culture supernatants via Protein-A and size exclusion chromatography. In cases where removal of the Fc portion was desired, the proteins were cleaved with Factor Xa (e.g., Factor Xa Protease HTI No. HCXA-0060 from Hematologic Technologies Inc.) according to the manufacturer's recommendations and subsequently purified by Protein-A and size exclusion chromatography. In cases where biotinylated proteins were desired, commercially available biotinylation kits were used (e.g., EZ-Link Amine-PEG3-Biotin from Pierce #21347) and the degree of biotinylation was characterized using commercially available kits (e.g., Biotin Quantitation Kit from Pierce #28005).

A single N-terminal domain 1 of human CEACAM6 was produced as a 6x His fusion protein construct in E. coli BL21 DE3 using the pET28a vector (Novagen). After overnight induction with IPTG at 37°C, the recombinant protein was isolated and refolded from inclusion bodies. Prior to refolding, the inclusion bodies were washed in Tris buffer, pH 8.5, containing 150 mM NaCl, 1 mM EDTA, and 0.1% Tween 20, and dissolved in the same buffer containing 8 M urea and no detergent. The solution was slowly diluted (1:10) into 50 mM CHES, pH 9.2, containing 500 mM arginine and incubated at 4°C for 16 hours. Purification was achieved by standard nickel-NTA chromatography and size exclusion chromatography on a Superdex 75 in 30 mM Tris buffer, pH 8.5, 150 mM NaCl.

The 9A6 murine IgG1 antibody (GM-0509) was obtained from Genovac and chimerized with human IgG1 and human IgG2. The Neo201 protein sequence, which served as either human IgG1 or human IgG2, was based on US20130189268. All antibodies were expressed in HEK293 cells using standard transient transfection procedures and purified from cell culture supernatants via protein-A and size exclusion chromatography.

Stable HeLa cell lines expressing different full-length human CEACAM receptors were generated. Thus, the following receptor sequences were transfected: human CEACAM1 (TPP-4185), human CEACAM3 (TPP-4187), human CEACAM5 (TPP-4188), human CEACAM6 (TPP-4639), human CEACAM8 (TPP-4190), human CEACAM19 (TPP-4186), or cynomolgus monkey CEACAM6 (TPP-4189). The HeLa cell line does not endogenously express any of these receptors on the surface, as confirmed by FACS analysis, and surface expression was only detected after transfection of the corresponding CEACAM receptor. Briefly, expression constructs were cloned into UCOE-based vectors (EMD Millipore Corporation) and transfected into HeLa cells. After selection with hygromycin, suitable stable clones were screened by Western blotting of total cell lysates and FACS staining of the cell surface using appropriate antibodies (human CEACAM1: #MAB22441 from R&D Systems; human CEACAM5: #MAB41281 from R&D Systems; human CEACAM6: #MAB3934 from R&D Systems; human CEACAM8: ab90294 from abcam; human CEACAM19: #NBP1-70494 from Novus; human CEACAM3: AF4166 from R&D Systems; cynomolgus monkey CEACAM6: Neo201-hIgG1).

Example 2: Characterization of the immunomodulatory 9A6-mIgG1 antibody

The 9A6 antibody has been described in the literature as immunomodulatory (Witzens-Harig et al., Blood 2013 May 30;121(22):4493-503). The antibody was characterized with respect to its affinity, its selectivity for other human CEACAMs, its cross-reactivity with monkey CEACAM6, its specific binding to a specific domain on CEACAM6, and its selectivity for other human CEACAMs.

Affinity measurement using surface plasmon resonance (SPR)

Surface plasmon resonance (SPR) experiments were performed using a Biacore T100, Biacore T200, or Biacore 4000 instrument (GE Healthcare Biacore, Inc.) equipped with a Series S sensor chip CM5 (GE Healthcare Biacore, Inc.). Binding assays were performed at 25°C using assay buffer HBS-EP+ (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20). Antibodies were captured using an anti-hIgG capture antibody covalently fixed to the chip surface via amine coupling chemistry. Amine coupling reagents (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), ethanolamine-HCl pH 8.5) were used from an amine coupling kit (GE Healthcare, product number BR-1000-50). Anti-hIgG and anti-mIgG capture antibodies from the Human Antibody Capture Kit (GE Healthcare, BR-1008-39) and Mouse Antibody Capture Kit (GE Healthcare, BR-1008-38), respectively, and immobilization buffer (10 mM sodium acetate, pH 5.0) were used. The sensor chip surface was activated by passing a freshly prepared solution of 0.2 M EDC and 0.05 M NHS over the chip surface at a flow rate of 10 µl/min for 420 seconds, followed by injection of anti-hIgG or anti-mIgG capture antibody (dissolved to 25 μg/ml in immobilization buffer) at a flow rate of 5 µl/min. Excess activated groups were blocked by injecting a 1 molar solution of ethanolamine at a flow rate of 10 µl/min for 420 seconds.

CEACAM antigen was used as the analyte to determine K _values . Antibodies were captured for 20 seconds at a flow rate of 10 μl/min before each analyte injection. For kinetic affinity determinations, various concentrations of human CEACAM1, human CEACAM3, human CEACAM5, human CEACAM6, cynomolgus CEACAM6, and cynomolgus CEACAM6-domain 1 protein (see above) between 1.56 and 200 nM in assay buffer were injected over the captured antibodies at a flow rate of 60 μl/min for 3 minutes, and dissociation was monitored for 10 minutes.

The obtained sensorgrams were double-referenced, i.e., online reference chamber correction, followed by buffer sample subtraction. KD values were calculated based on the ratio _of the dissociation rate constant ( kd ) and the association rate constant ( ka ), which were obtained by global fitting of _the sensorgrams _with a first- _order 1: ₁ Langmuir binding model implemented in the Biacore evaluation software package (Biacore T100/T200/4000 Evaluation Software, GE Healthcare Biacore, Inc.) .

Sandwich competition assay using SPR

Sandwich competition experiments by SPR were performed in a manner similar to that described above with minor modifications. First, each antibody to be analyzed was covalently immobilized on the sensor surface via amine coupling (see above for details). To examine whether different antibodies compete for binding to a specific CEACAM antigen, each antigen was captured by injection onto the immobilized antibody, followed by immediate injection of the second antibody to be tested for competition. If the second antibody bound to the antigen bound by the primary antibody (+), then neither antibody showed competition, and vice versa, if no binding was observed upon injection of the second antibody (-), then both antibodies competed for similar epitopes.

Domain mapping studies using ELISA

To elucidate specific epitope information, several chimeric domain constructs were designed, expressed, and purified. Briefly, the wild-type human CEACAM6 sequence was fused to the C-terminus of a human IgG1 Fc fragment, expressed in HEK293 cells, and purified from the supernatant via Protein-A and size exclusion chromatography (TPP-1790). To generate different domain chimeras, the human sequence of a single domain was replaced with the corresponding macaque sequence (F6YVW1) in Fc-fused human CEACAM6. This resulted in three different domain chimeras: hDom1-hDom2-mDom3 (TPP-1793), hDom1-mDom2-hDom3 (TPP-1792), and mDom1-hDom2-hDom3 (TPP-1791), along with a wild-type macaque Fc fusion construct (TPP-1306) as a control. In addition to the chimeras, single domain 1 of human CEACAM6 (TPP-1794) was produced from E. coli as described above.

Mapping of the domain specificity of antibodies using ELISA assays:

For ELISA analysis, Fc-fused domain chimeras and single domain 1 were coated onto Nunc MaxiSorb plates and blocked with SmartBlock solution. After incubation for 1 hour with a range of IgG concentrations (1 nM-1000 nM), the plates were washed with PBS/T. Analysis of IgG bound to CEACAM6 domain constructs was achieved by detection using an anti-human IgG (Fab specific)-peroxidase antibody (A0293, Sigma). Fluorescence detection was performed using AmplexRed (A12222, Invitrogen) following standard protocols.

ELISA-based binding assay

ELISA was used to characterize the binding of antibodies to various CEACAM paralogs and orthologs. Black 384-well plates were coated with 25 μl/well of various CEACAM protein preparations at 2 μg/ml in coating buffer (Candor) for 1 hour at 37°C. After washing once with PBS/0.05% Tween-20, the wells were blocked with 100% Smart Block (Candor) for 1 hour at 37°C. After washing three times with PBS/0.05% Tween-20, a dilution series of antibodies in PBS/0.05% Tween-20/10% Smart Block was added, ranging from 2 μg/ml to 2 ng/ml, and the plates were incubated at room temperature for 1 hour. After washing three times with PBS/0.05% Tween-20, the appropriate secondary antibody was added. For detection of proteins with human Fc, such as TPP-1173, anti-human IgG HRP (Sigma A0170) was used at a 1:10,000 dilution. In order to detect proteins such as TPP-1744 using mouse Fc, anti-mouse IgG HRP (ThermoScientific 31432) was used at a dilution of 1:10.000. PBS/0.05% Tween-20/10% Smart Block was used as dilution buffer. The plates were incubated at room temperature for 1 hour. After washing three times, the plates were developed with Amplex Red (Life Technologies) and fluorescence was read at an emission wavelength of 590 nm. EC ₅₀ values were calculated using GraphPad Prism 6.0 software using a four-parameter nonlinear curve fit.

result

To measure the monovalent affinity of 9A6 for human CEACAM6 and to evaluate its cross-reactivity to monkey CEACAM6, SPR experiments were performed as outlined above. The results are shown in Table 3:

Aliases Recombinant human CEACAM6 (R&D Systems; TPP-1436) Recombinant macaque CEACAM6 (TPP-1306) 9A6-mIgG1 TPP-1744 twenty two - Neo201-hIgG1 TPP-1173 10 twenty three

“-“: No binding detected

Table 3: SPR analysis: monovalent KD (in _nM ).

As shown, 9A6-mIgG1 can bind to recombinant human CEACAM6 with high affinity (22 nM). However, no binding to macaque CEACAM6 was detected. For comparison, Neo201-hIgG1 was also tested. This antibody showed high-affinity binding to both human and monkey CEACAM6. In summary, 9A6 showed high-affinity binding to human CEACAM6, but did not cross-react with monkey CEACAM6.

To map the binding domain of 9A6 on CEACAM6, binding to different human/monkey chimeras was evaluated by ELISA as outlined above. To enable comparison with Neo201-hIgG1 (TPP-1173), 9A6 was chimerized with human IgG1 (TPP-1745). The results are shown in Table 4:

TPP-# Origin of domain 1 Origin of domain 2 Origin of domain 3 9A6-hIgG1 (TPP-1745) Neo201-hIgG1 (TPP-1173) hWT 1790 people people people + + DOM1 MM 1791 macaques people people - + DOM2 MM 1792 people macaques people + + DOM3 MM 1793 people people macaques + + hDOM1 1794 people - - + - MM WT 1306 macaques macaques macaques - +

"+" indicates that binding was detected; "-" indicates that no binding was detected

Table 4: Domain mapping analysis by ELISA binding assay.

9A6 bound to wild-type human CEACAM6 as well as chimeras adopting domains 2 or 3 of macaque CEACAM6. However, it did not bind to chimeras adopting domain 1 of macaque CEACAM6 or to wild-type macaque CEACAM6. Consistent with this, it bound to single domain 1 of human CEACAM6. In contrast, Neo201-hIgG1 bound to all tested formats except single domain 1 of human CEACAM6. In summary, 9A6 binds to the N-terminal domain 1 of human CEACAM6.

To confirm the results, competition experiments were performed as outlined above. The results are shown in Table 5.

If the secondary antibody binds to the antigen bound by the primary antibody (+), then both antibodies show no competition, vice versa, if no binding is observed by injection of the secondary antibody (-), then both antibodies compete for similar epitopes

Table 5: Sandwich competition experiments by SPR for recombinant human CEACAM6 (R&D Systems, TPP-1436).

As is apparent from Table 5, 9A6-mlgG1 and Neo201-hIgG1 do not compete with each other for binding to human CEACAM6. This is consistent with the published epitope of Neo201 existing outside of domain 1.

In order to analyze the selectivity of 9A6 for different CEACAM6 orthologs and to allow comparison with Neo201-hIgG1, 9A6 was chimerized with hIgG1 (TPP-1745). In ELISA binding experiments performed as outlined above, the _EC50 values listed in Table 6 have been obtained:

“-“ indicates EC ₅₀ > 10 nM

Table 6: Selectivity/cross-reactivity analysis by binding ELISA - _EC50 values in nM.

9A6 was confirmed to bind to human CEACAM6 with high affinity. Consistent with SPR experiments using rhesus macaque CEACAM6, 9A6 also failed to bind to cynomolgus macaque CEACAM6. However, it was selective for CEACAM6, as no binding to human CEACAM3 or CEACAM5 was observed.

In contrast, Neo201-hIgG1 showed similar high-affinity binding to human CEACAM6 and even cross-reacted with cynomolgus monkey CEACAM6, resulting in reduced selectivity as it also showed high-affinity binding to human CEACAM5.

Example 3: CEACAM protein sequence alignment

The mature extracellular form of human CEACAM6 (amino acids 35-320 of UniProtKB/Swiss-Prot: P40199.3) consists of different domains: N-terminal domain 1 (amino acids 35-142 of UniProtKB/Swiss-Prot: P40199.3 according to www.uniprot.org), domain 2 (amino acids 145-232 of UniProtKB/Swiss-Prot: P40199.3 according to www.uniprot.org), and domain 3 (amino acids 237-314 of UniProtKB/Swiss-Prot: P40199.3 according to www.uniprot.org). Since the goal was to identify selective high-affinity antibodies to the N-terminal domain 1 of CEACAM6 (while cross-reacting with cynomolgus monkey CEACAM6), the possibility of combining the desired properties in a single molecule was evaluated.

To this end, the protein sequence of the N-terminal domain 1 of human CEACAM6 was compared with other proteins using the Blastp algorithm (NCBI) with standard settings to identify the most related CEACAM6 homologs. The (partial) mature extracellular domains of human CEACAM6 (amino acids 35-320 of UniProtKB/Swiss-Prot: P40199.3), human CEACAM1 (amino acids 35-428 of UniProtKB/Swiss-Prot: P13688.2), human CEACAM3 (amino acids 35-155 of UniProtKB/Swiss-Prot: P40198.2), human CEACAM5 (amino acids 35-417 of UniProtKB/Swiss-Prot: P06731.3), and cynomolgus monkey (Macaca fascicularis) CEACAM6 (amino acids 35-320 of TPP-4189) were aligned using "Global Alignment - Wilbur and Lipman (fast)" in the Phylospher software (Genedata). The alignment is shown in Figure 1. The percent sequence identity (based on alignment) of the N-terminal domain 1 of human CEACAM6 with other N-terminal domains was determined using Vector NTI software (Life Technologies). Those results are shown in Table 7.

Sequence identity of the N-terminal domain to the N-terminal domain 1 of human CEACAM6 Human CEACAM6 (100%) Human CEACAM3 90% Human CEACAM1 90% Human CEACAM5 89% Cynomolgus monkey CEACAM6 81%

Table 7: Percent protein sequence identity of the N-terminal domains of different CEACAMs with the N-terminal domain 1 of human CEACAM6.

The sequence alignment in Figure 1 shows a very high degree of similarity between the protein sequences of human CEACAM6 and those of human CEACAM3, human CEACAM5, and human CEACAM1 throughout the extracellular region. The target region (Domain 1 of human CEACAM6) is particularly similar to other CEACAMs, as also reflected in Table 7. Human CEACAM6 paralogs are much more similar to human CEACAM6 than the cynomolgus monkey ortholog. In fact, only two positions in the N-terminal region of the primary sequence are identical between human and cynomolgus monkey CEACAM6, but differ in the amino acids at those positions in other human paralogs (marked with asterisks in Figure 1).

It was concluded that the identification of high-affinity antibodies that are selective for the N-terminal domain 1 of human CEACAM6, but still cross-react with monkey CEACAM6, is highly challenging.

Example 4: Antibody Generation by Phage Display

To identify human anti-CEACAM6 antibodies, various selections were performed using the human Fab phage library FAB-300 from DYAX essentially as described previously (Hoet et al., 2005; Huang et al., 2006). As summarized in Table 8, up to four rounds of phage selection were performed using biotinylated, Fc-tagged recombinant CEACAM6 from humans and cynomolgus monkeys (TPP-1436 and TPP-2443) coated on streptavidin beads and the human tumor cell line KPL-4 (Kurebayashi et al., Br J Cancer. 1999 Feb;79(5-6):707-17), which expresses high levels of endogenous target protein on the cell surface. In addition, prior to each selection for the protein target, depletion of binders to CEACAM5 (TPP-1438; hC5) and CEACAM1 (TPP-1437; hC1) (non-target) or recombinant human IgG1-Fc (Fc) was included as indicated. For example, in strategy A, after depletion on human CEACAM1-coated beads (hC1), the first round of panning was performed on human CEACAM6 (hC6). The resulting output was split, and a portion was used for the second and third rounds of selection on human CEACAM6. Another portion was used for the second round of panning on KPL-4 cells, the third round of panning on human CEACAM6, and the final fourth round of panning on KLP-4 cells. In strategy C, a specific elution step was performed using mouse mAb 9A6-mIgG1 (TPP-1744).

Table 8: Phage selection strategy: human hC6 = TPP-1790; cynomolgus monkey cynoC6 = TPP-2443, hC1 = TPP-1438, hC5 = TPP-1437, Fc = recombinant human IgG1 Fc (R&D Systems #110-HG-100).

Phage pools enriched from different rounds of selection were screened for target and non-target binders by Fab-phage ELISA or by FACS analysis on CEACAM6-expressing cells as described (Hoet et al., Nat Biotechnol. 2005 Mar;23(3):344-8). Phage pools with favorable profiles were selected for gene III removal and subsequently ELISA-screened for soluble Fabs in ELISA. The DNA of the resulting sFab hits was sequenced, and cellular binding of unique representatives was characterized by FACS analysis on KPL-4 cells (Table 9). In some strategies, phage binders according to the invention were directly recloned into IgG.

Protein-ID (as hIgG1) FACS titration of sFab on KPL-4 cells TPP-1667 ++ TPP-1668 + TPP-1669 + TPP-1670 + TPP-1672 + TPP-1673 + TPP-1674 + TPP-1676 + TPP-1677 0 TPP-1678 0 TPP-1679 +++ TPP-1680 + TPP-1684 0 TPP-1686 +

+++: >1000 events at ~2.2 µg/ml sFab

++: >100 events at ~2.2 µg/ml sFab

+: >100 events at ~6.7 µg/ml sFab

0: Below the threshold

Table 9: FACS titration of unique sFab-hits:

The binding of phage display selected purified Fab fragments (see list in Table 9) to biotinylated variants of human CEACAM1 (TPP-1437), human CEACAM5 (TPP-1438), and human CEACAM6 (TPP-1436) was analyzed by biolayer interferometry using an Octet RED384 instrument (Pall ForteBio Corp.) The biotinylated antigens were loaded onto streptavidin (SA) biosensors (ForteBio Part No. 18-5019) and after a baseline equilibration step in assay buffer (PBS supplemented with 0.1% (w/v) BSA, 0.02% (v/v) Tween 20, and 0.05% (v/v) sodium azide; ForteBio Part No. 18-5032), the binding of Fab diluted to a final concentration of 200 nM in assay buffer was monitored for 300 seconds, followed by a 300 second dissociation phase.

The corresponding purified Fab fragments from Table 9 also showed binding to human CEACAM6, but not to human CEACAM5 or human CEACAM1.

To analyze whether Fabs compete with 9A6 for binding to human CEACAM6, a competition experiment was performed. Here, biotinylated human CEACAM6 (TPP-1436) was loaded onto the SA biosensor and the binding response of the Fab was compared to that obtained with 9A6-mIgG1 (TPP-1744) saturated loaded CEACAM6 (as outlined in Example 1). If the binding response in the presence of 9A6 is significantly reduced or abolished, this strongly suggests that the tested Fab binds to an epitope similar to that of 9A6.

Surprisingly, all tested Fabs were able to compete with 9A6 for binding to human CEACAM6.

The Fab sequences were reformatted into human IgG1 format for further characterization.

Similar to the experimental procedures described in Example 2, the affinity (monovalent KD ) of the reformatted antibodies for recombinant human CEACAM6 (TPP-1436) was determined by SPR. Sensorgrams were evaluated by global fitting of the sensorgrams using a first _- order 1:1 Langmuir binding model or using steady-state affinity analysis implemented in the Biacore evaluation software (Biacore T200/4000 Evaluation Software) package. The results are shown in Table 10:

Protein-ID TPP-1667 (750) TPP-1668 (550) TPP-1669 (185) TPP-1670 (340) TPP-1672 (580) TPP-1673 (515) TPP-1674 (600) TPP-1676 (1980) TPP-1677 (580) TPP-1678 (300) TPP-1679 76 TPP-1680 (870) TPP-1684 (480) TPP-1686 (310) TPP-2968 weak

Values in brackets: not accurately determined under the current experimental conditions, but sufficient for comparison between

Table 10: SPR analysis: monovalent KD (in _nM ).

As is apparent from Table 10, the antibodies exhibited rather low monovalent affinities, with the lowest value (highest affinity) being 76 nM for TPP-1679. The three IgGs exhibiting the highest monovalent affinities were analyzed in ELISA binding experiments (performed similarly to the protocol given in Example 2) with respect to their selectivity and cross-reactivity to cynomolgus monkey CEACAM6.

“-“ indicates EC ₅₀ > 10 nM (in the case of cynomolgus monkey CEACAM6, > 100 nM)

Table 11: Selectivity/cross-reactivity analysis by binding ELISA: _EC50 values in nM.

In summary, antibodies with rather poor monovalent affinities were obtained. This may be a trade-off, as binding to other paralogs is avoided. However, the selectivity profiles were generally insufficient (see TPP-1679 and TPP-1669 in Table 11). Of all the antibodies tested, TPP-1678 was the only one with very negligible cross-reactivity to recombinant cynomolgus monkey CEACAM6 (see Table 11).

In conclusion, no therapeutically useful anti-CEACAM6 antibodies were obtained using phage display (without further maturation).

Example 5: Antibody maturation of phage display-derived antibodies

To obtain antibodies with desired affinity, selectivity, and cross-reactivity profiles, some phage display-derived antibodies are affinity matured.

Therefore, all CDR amino acid positions of TPP-1669, TPP-1678, and TPP-1679 were individually randomized. The resulting variants were expressed and evaluated for binding to multiple CEACAM family members (human CEACAM6, cynomolgus monkey CEACAM6, human CEACAM3, and human CEACAM5) in cell supernatants by binding ELISA.

For TPP-1669, it was also not possible to identify individual mutations that enhance binding to cynomolgus monkey CEACAM6 while not enhancing binding to other human CEACAM family members.

For TPP-1679, several individual mutations that enhanced binding to human CEACAM6 without concomitant enhanced binding to other human CEACAM family members were identified, and a recombinant library containing all possible permutations was generated. The corresponding variants were expressed as human IgG2 isotype, purified, and evaluated for binding to multiple CEACAM family members by SPR similar to the experimental procedures described in Example 2.

Table 12 summarizes the properties of selected antibodies obtained by this method.

Human CEACAM6 (R&D Systems; TPP-1436) Cynomolgus monkey CEACAM6 (APP-319; (TPP-2443 cleaved) Human CEACAM5 (R&D Systems; TPP-1438) Human CEACAM3 (SinoBiological; TPP-2755) Human CEACAM1 (R&D Systems; TPP-1437) TPP-3399 9 - weak weak - TPP-3400 6 weak weak - - TPP-3401 16 - (94) - - TPP-3402 18 - (172) - - TPP-3403 13 weak (144) - - TPP-3404 11 (363) (138) - - TPP-3405 14 (522) (126) weak - TPP-3406 (15) - - - - TPP-3407 (12) - - weak - TPP-3408 (27) - - - -

Values in brackets: not accurately determined under current experimental conditions

“-“: No binding was detected under the current experimental conditions

“Weak”: if the two highest analyte concentrations analyzed (i.e., 100 and 200 nM) resulted in a binding signal between three times the signal-to-noise ratio and 20% of the theoretical maximal binding response (R max _theoretic )

Table 12: SPR analysis: monovalent KD (in _nM ).

The results obtained in Table 12 indicate that affinity and selectivity enhancement is quite possible. However, it also highlights that it is challenging to obtain cross-reactive binders to cynomolgus CEACAM6 that are at least within an order of magnitude close to the monovalent affinity for human CEACAM6.

For TPP-1678, several individual mutations that enhanced binding to human CEACAM6 and cynomolgus monkey CEACAM6 without a higher concomitant enhanced binding to other human CEACAM family members were identified, and recombinant libraries containing several permutations were generated. The corresponding variants were expressed as human IgG2 isotype, purified, and evaluated for binding to multiple CEACAM family members by SPR similar to the experimental procedures described in Example 2 (summarized in Table 13).

The binding characteristics of these antibodies were also determined by binding ELISA (monovalent binding, biotinylated CEACAM protein): black 384-well Maxisorp plates (Nunc) were coated with anti-human IgG (Sigma, I2136) at a 1:440 dilution in coating buffer (Candor) for 1 hour at 37°C. After washing once with PBS/0.05% Tween-20, the plates were blocked with 100% SmartBlock (Candor) for 1 hour at 37°C. After three washes, 2 µg/ml of the relevant antibody was added to the plates in PBS/0.05% Tween-20/10% SmartBlock. The plates were incubated at room temperature for 1 hour. After three washes, a dilution series of the relevant biotinylated CEACAM protein in PBS/0.05% Tween-20/10% SmartBlock was added, and the plates were incubated at room temperature for 1 hour. After three washes, 1 μg/ml streptavidin-peroxidase (Sigma, S5512) in PBS/0.05% Tween-20/10% SmartBlock was added and the plates were incubated at room temperature for 30 minutes. After three washes, the plates were developed with Amplex Red (Life Technologies) and fluorescence was read at an emission wavelength of 590 nm. _EC50 values were calculated using GraphPad Prism 6.0 software using a four-parameter nonlinear curve fit.

For selected antibodies obtained by this method, variants with significantly improved affinity, selectivity and cross-reactivity profiles were identified, as summarized in Tables 13 and 14.

Human CEACAM6 (R&D Systems; TPP-1436) Cynomolgus monkey CEACAM6 (APP-319; (TPP-2443 cleaved) Human CEACAM5 (R&D Systems; TPP-1438) Human CEACAM3 (SinoBiological; TPP-2755) Human CEACAM1 (R&D Systems; TPP-1437) TPP-3705 28 32 - - - TPP-3707 28 32 - - - TPP-3708 3 5 - weak (158) TPP-3709 8 20 - - (408)

Values in brackets: not accurately determined under current experimental conditions

“-“: No binding was detected under the current experimental conditions

“Weak”: if the two highest analyte concentrations analyzed (i.e., 100 and 200 nM) resulted in a binding signal between three times the signal-to-noise ratio and 20% of the theoretical maximal binding response (R max _theoretic )

Table 13: SPR analysis: monovalent KD (in _nM ).

TPP- Human CEACAM6 (R&D Systems TPP-1436 biotinylated) Cynomolgus monkey CEACAM6 (APP-319-biotinylated) Human CEACAM1 (R&D Systems TPP-1437 biotinylated) Human CEACAM3 (Sino Biological Inc. TPP-2755 biotinylated) Human CEACAM5 (R&D Systems TPP-1438 biotinylated) TPP-3705 0.18 0.12 113.04 - TPP-3707 0.19 0.11 139.18 - TPP-3708 0.02 0.03 9.64 1.04 - TPP-3709 0.04 0.07 36.53 4.68 - TPP-3470(9A6-hIgG2) 0.08 - - - -

“-“ indicates no detectable binding up to the highest concentration tested (150 ng/ml for human and cynomolgus monkey CEACAM6; 2000 ng/ml for human CEACAM1, human CEACAM3, and human CEACAM5)

Table 14: Selectivity/cross-reactivity analysis by binding ELISA: _EC50 values in nM.

In summary, using the TPP-1678 precursor, it was possible to obtain few high-affinity human antibodies to human CECACAM6 that truly cross-react with cynomolgus monkey CEACAM6 and are selective for CEACAM6: TPP-3707 binds approximately 730-fold better to human CEACAM6 than to human CEACAM3 (620-fold that of TPP-3705), as judged by comparison of their corresponding _EC50 values.

Example 6: Antibody Generation by Mouse Immunization

To generate mouse monoclonal antibodies against CEACAM6, two different immunization strategies were performed based on the sequences of the immunogens applied to Balb/c mice (Strategies A and B in Table 15). Within each strategy, mice were immunized with antigens applied via footpad or intraperitoneal injections over five rounds, as shown in Table 15. Strategy A focused on immunization with cynomolgus monkey CEACAM6-domain 1, while strategy B was based on a combination of full-length extracellular CEACAM6 from humans and cynomolgus monkeys as immunogens.

Immunization via the footpad was based on 5 weekly injections of 1 μg of antigen. Immunization via the intraperitoneal route was based on 4 IP injections biweekly (10 μg of antigen), followed by one booster by intravenous injection.

*: i.v. reinforcement

Table 15: Immunization schedule.

Four days after the last injection, lymph nodes or spleen cells of the mice were fused according to standard methods (e.g., Köhler and Milstein Nature. 1975 Aug7; 256(5517): 495-7). Screening of the resulting hybridoma clones was performed in an ELISA using biotinylated antigens and off-target proteins (as listed in Table 16). In more detail, microtiter plates were coated overnight with goat anti-mouse antibodies at 4°C. The next day, the plates were washed and blocked with 5% BSA for 2 hours at room temperature, followed by another washing step. 20 μl of hybridoma supernatant was incubated with biotinylated antigen at room temperature for 1 hour, and the mixture was transferred to the coated wells, followed by an incubation step (1 hour at room temperature). After washing the plates, anti-streptavidin-HRP conjugate was added at room temperature for 30 minutes. Finally, the wells were washed, and the color reaction was developed by adding 50 μl of TMB and recorded in a plate reader.

Target (all biotinylated) Non-target (all biotinylated) Human CEACAM-6 (TPP-1436) hCEACAM1 (TPP-1437) Cynomolgus monkey CEACAM6-Fc (TPP-2443) hCEACAM5 (TPP-1438) Cynomolgus monkey CEACAM6 (APP-319) Fc cleaved

Table 16: List of proteins used in ELISA-screening of hybridomas (target and non-target).

Surprisingly, only strategy A resulted in clones that displayed a favorable profile with respect to human and cynomolgus monkey CEACAM6 cross-reactivity and selectivity. In addition, some species-specific clones were obtained from both strategies.

Candidates positively selected by ELISA were subcloned through at least three cloning rounds and produced in larger quantities from ascites fluid by protein A chromatography.

Antibodies from mouse immunization were also characterized for binding to CEACAM6 in a cellular setting. HeLa cells overexpressing human or cynomolgus monkey CEACAM6 (see Example 1) were used in FACS experiments with supernatants or purified mIgs from hybridomas. Untransfected HeLa cells served as negative controls. Table 17 summarizes the profile of identified candidates from ELISA and FACS analysis:

Protein-ID Human CEACAM6 Cynomolgus monkey CEACAM6 TPP-2969 No binding detected Combine TPP-2970 No binding detected Combine TPP-2971 Combine Combine TPP-3100 Combine Combine TPP-3187 Combine Combine TPP-3101 Combine Combine TPP-3186 Combine Combine

Table 17: Summary of qualitative results of binding of murine hybridoma-derived antibodies to human and cynomolgus CEACAM6 from ELISA (using biotinylated TPP-1436, TPP-2443 and APP-319) and FACS-analysis (using transfected HeLa cells, see Example 1: TPP-4639 and TPP-4189).

The obtained murine antibodies were more closely characterized as purified mIgG by SPR analysis with respect to their monovalent _affinity ( KD ), their selectivity over other human paralogs and the extent of their cross-reactivity with cynomolgus monkey CEACAM6.

SPR was performed similarly to the experimental procedure described in Example 2.

The results are summarized in Table 18:

Human CEACAM6 (R&D Systems; TPP-1436) Cynomolgus monkey CEACAM6 (APP-319; (TPP-2443 cleaved) Cynomolgus monkey CEACAM6 domain 1 (APP-325; TPP-2452 cleaved) Human CEACAM5 (R&D Systems; TPP-1438) Human CEACAM3 (SinoBiological;TPP-2755) Human CEACAM1 (R&D Systems; TPP-1437) TPP-2969 72 (109) 51 - - - TPP-2970 72 56 47 - - - TPP-2971 61 25 30 - - - TPP-3100 79 n.t. 52 - - n.t. TPP-3101 69 n.t. 13 (300) weak n.t. TPP-3186 74 n.t. 53 - - n.t. TPP-3187 68 n.t. 42 - - n.t.

Values in brackets: not accurately determined under current experimental conditions

“-“: No binding was detected under the current experimental conditions

“Weak”: if the two highest analyte concentrations analyzed (i.e., 100 and 200 nM) resulted in a binding signal between three times the signal-to-noise ratio and 20% of the theoretical maximal binding response (R max _theoretic )

n.t. – Not tested

Table 18: SPR analysis: monovalent KD (in _nM ).

Unresolved differences were observed for TPP-2969 and TPP-2970: in initial ELISA and FACS analysis, they showed specificity for cynomolgus monkey CEACAM6, whereas in later SPR experiments, they also showed binding to recombinant human CEACAM6.

Summary: Immunization of mice with the cynomolgus macaque CEACAM6 N-terminal domain 1 (APP-325) surprisingly yielded some antibodies (e.g., TPP-3186, TPP-2971, TPP-3187) that were bona fide human-cynomolgus macaque CEACAM6 cross-reactive and at the same time selective for other human paralogs. The affinities were within acceptable ranges for therapeutic purposes, but their murine origin and associated immunogenicity precluded therapeutic application in humans.

Example 7: Antibody Humanization

To generate antibodies suitable for therapeutic use in humans, selected murine antibodies are humanized.

Select sequences of murine hybridoma-derived antibodies were determined by sequencing antibody cDNA from various hybridoma cell lines (see Table 17). Sequencing results revealed that TPP-3100 and TPP-3186 are identical. TPP-3101 produces a single heavy chain but two light chain sequences. TPP-2971 and TPP-3187 are highly similar, differing by four amino acids (see Figure 2).

The mouse VH and VL sequences of the humanized antibodies TPP-2971 and TPP-3187 were decrypted by transplanting the CDRs defined according to Kabat into the human germline framework. As an exception, HCDR2 was partially transplanted. Since the CDRs are very long (16 amino acids) according to the Kabat definition, only the first 9 amino acids were transplanted. These amino acids represent the portion of the HCDR2 identical to the HCDR2 defined according to the Chothia definition (for CDR definitions according to Kabat and Chothia, see: Andre C.R. Martin, "Proteinsequence and structure analysis of antibody variable domains", Antibody Engineering (Springer Lab Manuals), edited by Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg).

Human germline frameworks were selected based on similarity searches of the murine framework segments FW1, FW2, FW3, and FW4 to the human VH and VL collections and J element germline sequences. The murine CDRs were grafted into the best matching germline sequences (excluding the CDRs), which were IGKV1-9*01 and IGKJ2*01 for VL (identity 69.6%, FW1; 86.7%, FW2; 71.9%, FW3; 80.0%, FW4), and IGHV2-70*01 and IGHJ6*01 for VH (identity: 73.3% (TPP-2971) and 70.0% (TPP-3187), FW1; 85.7%, FW2; 71.9%, FW3; 90.9%, FW4). The germline sequences used in the similarity search were obtained from the VBASE2 dataset (Retter I, Althaus HH, Münch R, Müller W: VBASE2, an integrative V gene database. Nucleic Acids Res. 2005 Jan 1; 33(Database issue):D671-4). The names assigned to the most similar germline sequences were taken from the IMGT system (Lefranc, M.-P., Giudicelli, V., Ginestoux, C., Jabado-Michaloud, J., Folch, G., Bellahcene, F., Wu, Y., Gemrot, E., Brochet, X., Lane, J., Regnier, L., Ehrenmann, F., Lefranc, G. and Duroux, P. IMGT®, theinternational ImMunoGeneTics information system®. Nucl. Acids Res, 37, D1006-D1012 (2009); doi:10.1093/nar/gkn838)).

Two variants of the humanized sequence derived from TPP-2971 have been generated: TPP-3310 and TPP-3714. In the VH of TPP-3310, the J element was kept unchanged compared to the murine originator, while in the VH of TPP-3714, the J element was made to be completely human germline-like (see Figure 3). No glycosylation sites or unpaired cysteines were found in the humanized sequence.

In addition, two variants of the humanized sequence derived from TPP-3187 were generated: TPP-3820 and TPP-3821 (see Figure 4). Compared to TPP-3714, the VH of TPP-3820 contains a threonine instead of a serine at position 30 in HFW1, a glycine instead of an alanine at position 46 in HFW2, and two asparagine residues instead of two serine residues at positions 92 and 93 in LCDR3 in the VL. These four amino acid exchanges reflect the differences between the murine starting sequences of TPP-3187 and TPP-2971.

The variable domain VH of TPP-3821 is identical to that of TPP-3714, while the VL contains two asparagine residues instead of two serine residues in LCDR3 at positions 92 and 93, as compared to TPP-3714. These two amino acid exchanges reflect differences in the CDRs between the murine starting sequences of TPP-3187 and TPP-2971. No glycosylation sites or unpaired cysteine residues were found in the sequences of TPP-3820 and TPP-3821.

Affinity determination and sandwich competition experiments of chimeric and humanized antibodies were performed by SPR similar to the experimental procedures described in Example 2, and the results are summarized in Tables 19 and 20:

TPP-3308 (hIgG2 chimera of TPP-2971) TPP-3322 (hIgG2 chimera of TPP-3186) TPP-3323 (hIgG2 chimera of TPP-3187) TPP-3470 (hIgG2 chimera of 9A6) TPP-3308　　　　(hIgG2 chimera of TPP-2971) - - - - TPP-3322　　　　(hIgG2 chimera of TPP-3186) - - - - TPP-3323　　　　(hIgG2 chimera of TPP-3187) - - - - TPP-3470 (hIgG2 chimera of 9A6) - - - -

If the secondary antibody binds to the antigen bound by the primary antibody (+), then both antibodies show no competition, vice versa, if no binding is observed by injection of the secondary antibody (-), then both antibodies compete for similar epitopes

Table 19: Sandwich competition experiments by SPR for recombinant human CEACAM6 (R&D Systems, TPP-1436).

Human CEACAM6 (R&D Systems; TPP-1436) Cynomolgus monkey CEACAM6 (APP-319; (TPP-2443 cleaved) Human CEACAM5 (R&D Systems; TPP-1438) Human CEACAM3 (SinoBiological; TPP-2755) Human CEACAM1 (R&D Systems; TPP-1437) TPP-3310 13 31 - - - TPP-3714 13 27 - - - TPP-3820 27 54 - - - TPP-3821 twenty four 49 - - -

“-“ indicates that no binding was detected under the current experimental conditions

Table 20: SPR analysis: monovalent KD (in _nM ).

The selectivity and cross-reactivity analysis was performed by binding ELISA (monovalent, biotinylated CEACAM protein) analogously to the protocol provided in Example 5. The results obtained are summarized in Table 21.

TPP- Human CEACAM6 (R&D Systems TPP-1436 biotinylated) Cynomolgus macaque CEACAM6 (APP-319-biotinylated) Human CEACAM1 (R&D Systems TPP-1437 biotinylated) Human CEACAM3 (SinoBiological Inc. TPP-2755 biotinylated) Human CEACAM5　　　　(R&D Systems TPP-1438 biotinylated) TPP-3310 0.09 0.07 - - - TPP-3714 0.09 0.07 - - - TPP-3470 (hIgG2 chimera of 9A6) 0.08 - - - -

“-“ indicates no binding was detected at the highest concentration tested (150 ng/ml for human and cynomolgus monkey CEACAM6; 2000 ng/ml for human CEACAM1, human CEACAM3, and human CEACAM5)

Table 21: Selectivity/cross-reactivity analysis by binding ELISA: _EC50 values in nM.

The results in Table 19 indicate that TPP-2971, TPP-3186, and TPP-3187 compete for the same or similar epitopes on human CEACAM6 as 9A6-hIgG2. The results in Tables 20 and 21 highlight high affinity binding to human and cynomolgus monkey CEACAM6 with true cross-reactivity while being selective for CEACAM6 and not binding to CEACAM6 paralogs.

In conclusion, humanization was completely successful, with the antibody exhibiting higher affinity than its murine precursor, enabling therapeutic application in humans.

Example 8: Selective CEACAM6 binding on cells

To demonstrate the binding and selectivity of the anti-CEACAM6 antibodies to the authentic antigen, the antibodies were tested for binding to native CEACAM6 on the cell surface of different cell lines by FACS experiments.

CEACAM6 selectivity was tested on a panel of HeLa cells transfected with different CEACAM receptors (human CEACAM1, human CEACAM3, human CEACAM5, human CEACAM6, human CEACAM8, human CEACAM19, and cynomolgus monkey CEACAM6 - see Example 1) compared to binding to HeLa wild-type cells, which were negative for CEACAM6. _EC50 values were determined for binding to HeLa cells transfected with human and cynomolgus monkey CEACAM6. The results are shown in Table 22.

For FACS experiments, HeLa wild-type cells were cultured in RPMI-1640 with 10% FCS, while CEACAM receptor-transfected HeLa cells additionally received 0.5% gentamicin (stock 10 mg/ml, Fa.PAA) and 200 μg/ml hygromycin B (stock 50 mg/ml, Invitrogen). Cells were washed three times with PBS (Ca2 ⁺ /Mg2 ⁺ -free) and non-enzymatically detached from the culture plates using EDTA dissociation buffer (Gibco). Cells were washed in cold FACS buffer (PBS without Ca2 ⁺ /Mg2 ⁺ and heat-inactivated 3% FCS) and counted using a Countess machine (Invitrogen). ¹⁰⁵ cells/well were plated and incubated with the corresponding primary antibody (5 μg/ml) for 1 hour at 4°C on a plate shaker. The cells were then washed twice with FACS buffer (400 g, 5'), resuspended in 100 μl of secondary antibody (PE-anti-mouse or anti-human IgG, 1:150 dilution, Dianova #115-115-164, #109-115-098), and incubated for another hour on a plate shaker at 4° C. After washing twice, the cells were resuspended in 100 μl of FACS buffer and analyzed on a FACS Canto II machine (Beckton Dickinson) or FACS Array (Beckton Dickinson).

For _EC50 analysis, primary antibodies were used at increasing concentrations ranging from 0.1 nM to 100 nM. Half-maximal binding values ( _EC50 ) were determined by plotting median fluorescence intensity signals against concentration (logarithmic scale). Curve fitting of the data was performed using Graph Pad prism analysis software.

Definitions of -, +, ++, +++ as determined from the FACS median fluorescence log shift: - = no shift; + = log shift compared to control antibody, median 10-100; ++ = 2 log shift, median 100-1,000; +++ = 3 log shift, median 1,000-10,000

Table 22: Specific binding to a panel of CEACAM-receptor transfected HeLa cell lines.

The binding of anti-CEACAM6 antibodies to different cancer cell lines endogenously expressing CEACAM6 was also tested by FACS analysis. The cell lines were cultured according to the protocol provided by the American Tissue Culture Collection (ATCC). The observed binding signal was specific, as the non-binding isotype control did not cause a shift in the fluorescence signal. The half-maximal binding (EC50) values were in the low nanomolar range (Table 23).

*All cell lines were obtained from public tissue banks such as the American Tissue Culture Collection, except for the KS breast cancer cell line which was a gift from Dr. Brigitte Gückel (University of Tübingen).

Table 23: Binding of anti-CEACAM6 antibodies to endogenous CEACAM6-positive tumor cell lines ( _EC50 values).

In summary, selective binding to authentic human cell surface CEACAM6 (without binding to other human paralogs) was demonstrated for the murine TPP-2971, TPP-3100, TPP-3186, and TPP-3187 antibodies, as well as the human TPP-3820, TPP-3821, TPP-3310, TPP-3714, and TPP-3707 antibodies. Binding of these antibodies to human CEACAM6 was comparable to that of 9A6 on human CEACAM6-expressing cell lines. For TPP-3310, similar binding to endogenously expressed CEACAM6 on human tumor cell lines was demonstrated as for 9A6-hIgG2.

The antibodies of the invention also bind to cynomolgus monkey CEACAM6 with an affinity comparable to that of the human receptor, with binding _EC50s ranging from single digits to subnanomolar, while no binding of 9A6 to cynomolgus monkey CEACAM6 on the cell surface was detected at concentrations up to 100 nM. This result indicates that the antibodies of the invention truly cross-react with human and cynomolgus monkey CEACAM6, with 9A6 clearly binding preferentially to human CEACAM6.

Example 9: Thermal Stability Analysis

Use differential scanning calorimetry (DSC), use the VP-capillary DSC system (MicoCal Inc.) that cell volume is 0.137mL to study the thermal stability of IgG.All samples are diluted to the ultimate concentration of 0.5mg/mL in DPBS pH.7.4, and use the buffer control without protein as reference.Sample is scanned from 20 ℃ to 120 ℃ with a scanning rate of 120 ℃/h.By subtracting the buffer control scanning to correct the thermal analysis diagram gained, and use Origin 7.0 data analysis (OriginLab Corp.) to carry out homogenization for protein concentration.By fitting the DSC data to the nonlinear regression program (" Non-2-state:Cursor init ") provided with Origin, obtain melting temperature.

Thermal stability of the Fab domain TPP-3310 88.2 ℃ TPP-3714 88.5℃ * / 95.1℃ * TPP-3400 74.7 ℃ TPP-3707 80.6 ℃ TPP-3470 (9A6-hIgG2) 80.2 ℃

* Fab unfolds non-cooperatively

Table 24: Thermal stability of Fab domains.

All IgGs tested exhibited high thermal stability within the Fab domain. The thermal stability of TPP-3310 and TPP-3714 was significantly higher, far exceeding that of TPP-3470 (9A6-hIgG2). This is surprising, as high stability has been associated with antibodies with a VH3 framework (Honegger et al., 2009, Protein Eng Des Sel. 22(3):121-134).

The high thermal stability indicates that TPP-3310 and TPP-3174 have better drug suitability (better stability, less aggregation tendency, less immunogenicity risk) than TPP-3470 (9A6-hIgG2).

Example 10: Interference with the interaction between CEACAM6 and CEACAM1

It has been hypothesized that CEACAM1 may be a binding partner of CEACAM6 in trans on activated T cells (Witzens-Harig et al., Blood 2013 May 30;121(22):4493-503): Trans- and cis-interacting homophilic and heterophilic interactions between CEACAMs have been described, for example, between CEACAM1 and CEACAM5 or CEACAM6 and CEACAM8. CEACAM1 is displayed on activated T cells, where CEACAM1 ligation and phosphorylation recruit SH2 domain-containing protein tyrosine phosphatase 1 (SHP1). SHP1 dephosphorylates ZAP70, leading to inhibition of TCR signaling. Thus, CEACAM1 ligation leads to early inhibition of T cell activation within 10 minutes after activation.

Furthermore, a role for CEACAM5 in suppressing natural killer (NK) cell responses against colorectal cancer cells was reported (Zheng et al., PLoS One. 2011;6(6):e21146), which may be based on its heterophilic binding to inhibitory CEACAM1 expressed on NK cells.

Therefore, direct CEACAM6-CEACAM1 interactions were tested using recombinant proteins in a binding ELISA. After establishing in preliminary experiments that a moderate, but specific, interaction between CEACAM1 and CEACAM6 could be detected, the following protocol was used: Black 384-well Maxisorb plates (Nunc) were coated with 1 µg/ml CEACAM1 (R&D Systems, TPP-1437) in coating buffer (Candor) for 1 hour at 37°C or left uncoated as a control. After washing once with PBS/0.05% Tween-20, the wells were blocked with 100% SmartBlock (Candor) for 1 hour at 37°C. In a separate plate, a dilution series of the antibody of interest in PBS/0.05% Tween-20, 10% SmartBlock, was incubated with 2 µg/ml CEACAM6-Fc (TPP-1790) for 1 hour at room temperature. The blocked plates were washed three times, and the preformed antibody-CEACAM6 complexes were added. The plates were incubated for 1 hour at room temperature. After three washes, anti-human IgG HRP (Sigma A1070) was added at 1:10.000 and the plate was incubated for 1 hour at room temperature. After three washes, the plate was developed with Amplex Red (Life Technologies) and fluorescence was read at an emission wavelength of 590 nm.

“+” indicates competition

“-“ means non-compete

Table 25: Competition ELISA of antibodies competing for binding of human CEACAM6-Fc (TPP-1790) to passively coated human CEACAM1 (TPP-1437).

As shown in Table 25, it was possible to compete for the interaction of CEACAM6 with CEACAM1 with all antibodies tested except TPP-3688 (Neo201-hIgG2).

Taken together, this observation is consistent with the following hypotheses: a) CEACAM1 on activated T cells is a possible interaction partner of CEACAM6, leading to T cell inhibition, b) the N-terminal D1 domain of CEACAM6 is involved in the interaction between CEACAM1 and CEACAM6, and c) the antibodies of the present invention are able to interfere with the CEACAM6-CEACAM1 interaction.

Example 11: In vitro inhibition of the immunosuppressive activity of CEACAM6

The immunosuppressive function of CEACAM6 on tumor cells was recently investigated in vitro (Witzens-Harig et al., Blood 2013 May 30;121(22):4493-503) and in vivo (Khandelwal et al., Poster Abstract 61, Meeting Abstract from 22nd Annual International Cancer Immunotherapy Symposium October 6-8, 2014, New York City, USA). The commercially available 9A6 antibody (Genovac/Aldevron) was shown to inhibit the immunosuppressive activity of CEACAM6, resulting in enhanced cytokine secretion by T cells in vitro and antitumor efficacy in vivo.

In order to study the effect of the antibody of the present invention on the immunosuppressive activity of CEACAM6, a co-culture experiment of a model tumor cell line and a model tumor antigen-specific T cell clone was performed:

Tumor antigen-specific T cells were generated by the procedure described in Brackertz et al. (Brackertz et al., Blood Cancer J. 2011 Mar; 1(3): e11). Briefly, survivin-specific CD8+ T cells were isolated from peripheral mononuclear cells via CD8-specific magnetic activated cell sorting. Isolated HLA-A2 ^- CD8 ⁺ T cells were repeatedly stimulated with allogeneic HLA-A2 ⁺ dendritic cells loaded with 10 μg of the HLA-restricted peptide epitope survivin _95-104 (ELTLGEFLKL). After stimulation, proliferating T cells were ^stained with HLA-A2/survivin _95-104 multimers (A*02:01 391 LMLGEFLKL survivin 96-104, labeled with APC, ProImmune Limited, #F391-4A-E), FACS sorted, and cloned by limiting dilution in 96-well plates.

T cell clones were expanded by culturing 2 x 10 5 T cell clones in 40 ml of _RPMI -1640 medium with glutamine (Sigma-Aldrich), 10% human serum (human AB serum, Valley Biomedical, Inc, #HP1022), 1% penicillin/streptomycin (Life Technologies) at 37°C and ⁵ % CO 2 and feeder cells consisting of 5 x 10 ⁷ irradiated PBMCs (30 Gy) and 1 x 10 ⁷ irradiated LCLs from different donors (these B lymphoblastoid cell lines were generated by EBV transduction of peripheral blood B cells from healthy donors and an EBV-transfected monkey cell line (B95/8, ATTC) as described in Brackertz et al., Blood Cancer J. 2011 Mar;1(3):e11 and Dissertation Andreas Moosmann, Ludwig-Maximilians-University Munich, Germany, 2002) (100-150 Gy). This expansion was carried out for 14 days in the presence of 50 U/ml IL-2 (Proleukin, Novartis, #1003780), 2.5 ng/ml IL-15 (rhIL-15-CF R&D #247_IL-025/CF) and 30 ng/ml anti-human CD3 antibody (OKT3 eBiosciences 16-0037-85). The KS human breast cancer cell line (obtained from Dr. Brigitte Gückel (University of Tübingen, Germany)) was cultured in DMEM (Sigma-Aldrich) with 10% FCS (FBS Superior, Biochrom) and 1% penicillin/streptomycin at 37°C and 5% _CO2 .

To analyze the modulatory activity of anti-CEACAM6 antibodies on the immunosuppressive function of CEACAM6 in vitro, survivin peptide-specific CD8 ⁺ T cell clones were co-cultured with the CEACAM6 ⁺ , HLA-A2 ⁺ , and survivin ⁺ human breast cancer cell line KS, and IFN-γ secretion as a readout of T cell activity was measured by IFN-γ ELISpot or IFN-γ ELISA.

For co-culture, KS tumor cells were non-enzymatically dissociated using PBS-EDTA for 5 minutes, centrifuged, washed, and counted. The cell concentration was adjusted to 1 x 10 ⁵ cells/ml in X-Vivo-20 (Lonza), and cells were pretreated with anti-CEACAM6 antibody or isotype-matched control antibody on ice for 10 minutes. After the incubation step, 10,000 KS target cells were directly plated in triplicate onto IFN-γ-ELISpot or U-96-well ELISA plates. Simultaneously, survivin peptide-specific T cells were harvested, washed with X-Vivo-20, and plated onto KS target cells at the indicated cell numbers. Co-cultures of tumor cells, anti-CEACAM6 antibody, and T cells were incubated at 37°C for 20–40 hours. IFN-γ ELISpot plates (MABTECH: ELISpot assay for human interferon gamma #3420-3PT, mAB1-D1K anti-IFGg antibody, mAB7-B6-1-biotin, streptavidin-ALP, BCIP/NBT plus substrate for ELISpot #3650-10) and IFN-γ-ELISA (BD Human IFN-γ ELISA Set #555142) were developed according to the manufacturer's instructions. ELISpot plates were counted using a CTL ELISpot plate reader and the optical density of ELISA plates was measured using a Tecan Infinite M200 plate reader. The experiment was considered valid if the positive control, TPP-3470 (9A6-hIgG2), was statistically significant compared to the isotype-matched antibody control.

Co-culture of KS tumor cells with survivin-peptide-specific CD8 ⁺ T cells in the presence of anti-CEACAM6 antibody resulted in a statistically significant increase in IFN-γ production by T cells compared to samples not treated with anti-CEACAM6 antibody or treated with isotype-matched control antibody ( FIG. 5 ).

In conclusion, the cynomolgus monkey cross-reactive antibodies TPP-3310, TPP-3707, and TPP-3323 were able to alleviate CEACAM6-mediated immunosuppression of tumor antigen-specific T cells to the same extent as TPP-3470 (9A6-hIgG2), as measured by IFN-γ secretion by survivin peptide-specific CD8 ⁺ T cells or the number of IFN-γ-secreting activated T cells.

Example 12: Analysis of cytokine/chemokine profiles secreted by T cells treated with anti-CEACAM6 antibodies

To investigate the effects of anti-CEACAM6 antibodies on the cytokine/chemokine profile of human T cells with respect to improved cytotoxicity and potent anti-tumor immune responses, Luminex-based multiplex cytokine analysis of co-culture experiments of model tumor cell lines and model tumor antigen-specific T cell clones was performed.

Survivin peptide-specific CD8 ⁺ T cell clones were generated and expanded in vitro as described in Example 11. Tumor cell culture and ELISA co-cultivation were performed as described in Example 11.

After 20 hours of co-cultivation, the plates were centrifuged at 1400 rpm for 10 minutes and the supernatants were collected. Multiplex analysis was performed using the MILLIPLEX Human Cytokine/Chemokine Magnetic Bead Set - Premixed 38 Plex Analytes (Merck Millipore #HCYTMAG-60K-PX38) on a BioPlex100 system (Bio-Rad) according to the manufacturer's instructions. Standard curves and concentrations were calculated using Bio-Plex Manager 6.0. The experiment was considered valid if the positive control TPP-3470 (9A6-hIgG2) increased >1.5-fold compared to the isotype-matched antibody control.

Blockade of CEACAM6 by antibodies of the invention increased IFN-γ, IL-2, and TNF-α secretion by >1.5-fold in co-culture of survivin-peptide-specific T cells with KS tumor cells compared to control samples treated with isotype-matched controls ( FIG6 ).

In conclusion, the cynomolgus monkey cross-reactive antibodies TPP-3310 and TPP-3707 were able to shift the cytokine profile of survivin-peptide-specific CD8 ⁺ T cells to the same extent as TPP-3470 (9A6-hIgG2) towards a more cytotoxic and activated phenotype, characterized by increased IFN-γ, IL-2, and TNF-α secretion as measured by Luminex-based multiplex analysis.

Example 13: Antitumor Efficacy in the Adoptive T Cell Transfer KS Model

The antitumor efficacy of an anti-CEACAM6 antibody (9A6; Genovac/Aldevron) has been studied in an adoptive human T-cell transfer system, in which tumor-antigen-specific human T cells are expanded in vitro and co-injected with anti-CEACAM6 antibody into nude mice bearing human xenograft tumors (Khandelwal et al., Poster Abstract 61, Meeting Abstract from 22nd Annual International Cancer Immunotherapy Symposium October 6-8, 2014, New York City, USA).

In order to study the effect of the antibodies of the present invention on the anti-tumor efficacy, the following adoptive T cell transfer experiments were performed:

Survivin-peptide-specific CD8 ⁺ T cell clones were generated and expanded in vitro as described in Example 11.

Female NOD-Scid mice (NOD.CB17-Prkdc ^scid /J; Charles River, France) aged 6 to 8 weeks were injected subcutaneously with 2 x 10 ⁶ KS tumor cells (see Example 11). Mice were randomized on day 16, and mice with tumors less than 40 mm ² in surface area were excluded (= no tumor was harvested). Mice (n = 8-10 mice per group) were treated with iv adoptive transfer of 5 x 10 ⁶ survivin-peptide-specific T cell clones on days 23 and 27. 200 μg of anti-CEACAM6 antibodies TPP-3740, TPP-3707, TPP-3310, or their respective isotype-matched control antibodies were administered ip on days 22, 24, 26, and 28. A control group was injected with PBS instead of T cells and antibodies. Subcutaneously grown tumors were measured with a caliper, and the surface area was calculated using the formula "length x width." Only experiments in which the vehicle-treated control group of mice showed a stable and significant increase in tumor surface and tumor volume throughout the duration of the study were considered valid. The results of the experiments may have been influenced by parameters that are difficult to control: not only has the in vivo growth of the KS cell line proven to be variable, but also the survival of human T cells in mice and the infiltration of T cells into tumors show considerable variability.

Adoptive transfer of survivin-peptide-specific T cells in combination with anti-CEACAM6 antibodies resulted in a reduction in tumor burden compared to T cells injected with matched isotype control or PBS control groups (Figure 7). Similar efficacy was observed using TPP-3740 (9A6-hIgG2).

In conclusion, in an adoptive T cell transfer model using survivin-peptide-specific CD8 ⁺ T cells and KS tumors, the cynomolgus monkey cross-reactive antibodies TPP-3310 and TPP-3707 exhibited antitumor efficacy to the same extent as TPP-3470 (9A6-hIgG2).

Example 14: CEACAM6-positive tumor cell lines and tumor tissues

CEACAM6 is expressed in various cancers, which are potential target indications for treatment with CEACAM6 immunomodulatory antibodies. Therefore, cancer cell lines from different origins and representing different cancers were tested for CEACAM6 expression by FACS analysis. The results are shown in Table 26.

Cancer cell lines obtained from public tissue banks such as the American Tissue Culture Collection (ATCC) are cultured according to the supplier's instructions.

Cells were washed three times with PBS (Ca ²⁺ /Mg ²⁺ -free) and non-enzymatically detached from the culture plate using EDTA dissociation buffer (Gibco). Cells were washed in cold FACS buffer (PBS without Ca ²⁺ /Mg ²⁺ and heat-inactivated 3% FCS) and counted using a Countess cell counter (Invitrogen). 10 ⁵ cells/well were plated and incubated with mouse monoclonal antibody 9A6 (TPP-1744; 5 µg/ml) or purified NA/LE mouse IgG1 isotype control antibody (BD Pharmingen #553447) for 1 hour at 4°C on a plate shaker. The cells were then washed twice with FACS buffer (400 g, 5'), resuspended in 100 μl of PE-labeled anti-mouse secondary antibody (1:150 dilution, Dianova #115-115-164), and incubated on a plate shaker at 4°C for another hour. After washing twice, the cells were resuspended in 100 μl of FACS buffer and analyzed on a FACS Canto II machine (Beckton Dickinson) or FACS Array (Beckton Dickinson). The observed binding signal was specific because the non-binding isotype control did not cause a shift in the fluorescence signal (Table 26).

Cell line* Sources of cancer type CEACAM6　　　　　Surface expression MCF7 breast Adenocarcinoma + MCF7/AdrVp (MDR breast CA) breast Adenocarcinoma + KS breast Malignant overflow, breast cancer patients +++ BT-20 breast Adenocarcinoma, ER-negative - BT-474 breast Ductal carcinoma; +++ HCC38 breast Primary ductal carcinoma - MDA-MB-361 breast Adenocarcinoma, originating from metastatic site: brain +++ MDA-MB-453 breast Metastatic cancer, arising from metastatic site: pericardial effusion - MFM-223 breast Epithelioid, ductal carcinoma +/- MX-1 breast Invasive ductal carcinoma, ER-negative - SUM 149 breast Invasive ductal carcinoma, ER-PR-inflammatory breast cancer - SUM 159 breast Er-, PR-anaplastic breast cancer - T-47D breast Ductal carcinoma, from metastatic sites: pleural effusion + ZR-75-1 breast Ductal carcinoma, from metastatic sites: ascites ++ HPAC pancreas Adenocarcinoma ++++ SW1990 pancreas Adenocarcinoma, from metastatic site: spleen - PaTu 8902 pancreas Adenocarcinoma +-++ AsPC-1 pancreas Adenocarcinoma, from metastatic site: ascites +++ DAN-G pancreas Adenocarcinoma + MIA PaCa-2 pancreas - Panc1 pancreas Ductal, epithelioid pancreatic cancer -　　　　　　　　　　　　　- BxPc3 pancreas Adenocarcinoma +++ Capan-2 pancreas Adenocarcinoma - PC3.gd.neo (PaCa) prostate Adenocarcinoma, from metastatic site: bone - HPAFII (PaCa) pancreas Adenocarcinoma +++ SW 1463 rectum Epithelial, Dukes C, colorectal adenocarcinoma +/- SNU-C1 colon Adenocarcinoma, from metastatic site: peritoneum ++ HT55 colon epithelium +++ Colo 201 colon Dukes D type, adenocarcinoma, from metastatic site: ascites - Colo320DM colon Dukes D type, colorectal adenocarcinoma - CaCo-2 colon Adenocarcinoma +-++ SW403 colon Dukes C adenocarcinoma - HCC2998 colon Adenocarcinoma ++ RKO colon - KM-12 colon Adenocarcinoma ++ CL-34 the large intestine Adenocarcinoma + COLO 205 colon Dukes D type, adenocarcinoma '+/- HCT 116 colon - SW480 colon Duke B type, colorectal adenocarcinoma -　　　　　　　　　　- WiDr colon Adenocarcinoma ++ DLD-1 colon Dukes C type, adenocarcinoma - SW 620 colon Dukes C type, adenocarcinoma, from metastatic site: lymph nodes - SW1116 colon Adenocarcinoma +++ T-84 colon Colonic metastasis from the lungs + - ++ LoVo colon Adenocarcinoma ++ HCT15 colon Dukes C type, adenocarcinoma - HT29 colon Adenocarcinoma ++ LS174T colon Dukes B type, adenocarcinoma +/- EKVX lung Adenocarcinoma - A549 lung +++ NCI-H1688 lung SCLC + Calu-1 lung Epidermoid carcinoma, K-ras positive - Calu-3 lung Adenocarcinoma + HCC827 lung Adenocarcinoma ++ LXF-289 lung Adenocarcinoma - NCI-H1299 lung From metastatic sites: Lymph nodes - NCI-H1437 lung Adenocarcinoma, from a metastatic site. Pleural effusion +++ NCI-H146 (SCLC) lung From metastatic site (bone marrow) +/- NCI-H1581 lung Large cell NSCLC - NCI-H1975 lung Adenocarcinoma - NCI-H1993 lung Adenocarcinoma, from metastatic site: Lymph nodes +++ NCI-H2228 lung Adenocarcinoma ++ NCI-H226 lung Squamous cell carcinoma, mesothelioma, from metastatic sites: pleural effusion - NCI-H23 lung Adenocarcinoma - NCI-H292 lung Mucoepidermoid lung cancer - NCI-H322 lung Bronchoalveolar carcinoma - NCI-H358 lung Bronchoalveolar carcinoma, arising from metastatic alveoli - NCI-H441 lung Papillary adenocarcinoma ++ NCI-H460 lung Large cell lung cancer, from metastatic sites: pleural effusion - NCI-H520 lung squamous cell carcinoma - NCI-H522 lung Adenocarcinoma K-ras, mutant p53 - NCI-H661 lung Large cell lung cancer, from metastatic sites: lymph nodes - NCI-H69 (SCLC) lung - NCI-H82 (SCLC) lung From metastatic sites: pleural effusion - SW 900 lung squamous cell carcinoma - HCC-2935 lung Adenocarcinoma ++++ HCC-1395 lung Primary ductal carcinoma - RPMI-8226 Myeloma Plasma cell carcinoma; Multiple myeloma (IgG lambda type) B lymphocytes; Lymphoblasts + SKMM2 Myeloma - L-363 Myeloma Plasma cell leukemia - JJN-3 Myeloma Plasma cell leukemia - KMS-12-BM Myeloma - KMS-12-PE Myeloma - LP-1 Myeloma - MOLP-2 Myeloma MOLP-8 Myeloma - SNU-1 Stomach From metastatic site : ascites +++ Hela cervix Adenocarcinoma - DMS-153 (SCLC) lung From metastatic site: Liver - MeWo skin Fibroblasts, malignant melanoma, from metastatic sites: lymph nodes -

Definitions of -, +, ++, and +++ as determined from the FACS median fluorescence log shift: - = no shift; + = log shift compared to control antibody, median 10-100; ++ = 2 log shift, median 100-1,000; +++ = 3 log shift, median 1,000-10,000; ++++ = 4 log shift; median > 10,000

*All cell lines were obtained from public tissue banks such as the American Tissue Culture Collection, except for the KS breast cancer cell line which was a gift from Dr. Gueckel (Tübingen); B. Gueckel, Cancer Cell International 2004, 4(Suppl 1):S38).

Table 26: Results of the binding of the CEACAM6-specific antibody 9A6 mIgG1 (TPP-1744) and hence CEACAM6-expressing human cancer cell lines screening by FACS.

In summary, CEACAM6 is expressed in cell lines representing various cancers (e.g., colorectal cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), pancreatic cancer, gastric cancer, breast cancer, and multiple myeloma), which constitute potential target indications for treatment with CEACAM6 immunomodulatory antibodies and other response modifiers (e.g., peptides, small molecules, artificial scaffold binders, etc.).

Example 15: Binding to Single Domain 1 of Human and Cynomolgus Monkey CEACAM6

To test whether the antibodies of the present invention can bind to the isolated single domain 1 of human and cynomolgus monkey CEACAM6, SPR experiments were performed as in Example 1.

As described in Example 1, a single N-terminal domain 1 of cynomolgus monkey CEACAM6 (TPP-2453) was generated similarly to TPP-1794:

name Protein-ID describe SEQ-ID Cynomolgus monkey CEACAM6-domain 1-His TPP-2453 Domain 1, fusion with His (expressed in E. coli) SEQ-ID NO: 180

The affinity (monovalent K _D ) of the antibodies of the invention for the recombinant single domain 1 of human and cynomolgus monkey CEACAM6 was determined by SPR similarly to the experimental procedure described in Example 1 and is shown in Table 27.

Testing antibodies Isotype TPP-2971 Mouse IgG1 11 5.3 TPP-3186 Mouse IgG1 15 8.7 TPP-3187 Mouse IgG1 12 6.9 TPP-3308 Human IgG2 chimera 9.5 5.4 TPP-3310 Human IgG2 3.7 3.4 TPP-3322 Human IgG2 chimera 12 8.8 TPP-3323 Human IgG2 chimera 9 6.9 TPP-3705 Human IgG2 6.7 2.7 TPP-3707 Human IgG2 6.4 2.4 TPP-3714 Human IgG2 3.7 3.2 TPP-3820 Human IgG2 5.1 5.2 TPP-3821 Human IgG2 4.5 4.7 TPP-1745 Human IgG1 3.3 -

“-“: No binding detected

Table 27: SPR analysis: monovalent KD (in _nM ).

In conclusion, the antibodies of the present invention bind to the human and cynomolgus monkey N-terminal domain 1 of CEACAM6 with comparable affinity.

Example 16: X-ray crystal structure of a single N-terminal domain 1 of human CEACAM6 in complex with the Fab fragment APP-1574

The crystal structure of a single N-terminal domain 1 of human CEACAM6 (TPP-1794; SEQ ID NO 169) bound to a Fab fragment related to TPP-3310 (referred to as APP-1574) was determined.

To facilitate the preparation of Fab fragments, TPP-3310 was generated as a human IgG1 variant (designated TPP-5468, see Table 28). Papain cleavage of TPP-5468 and subsequent purification resulted in APP-1574. This Fab fragment contains the variable domains (VH and VL) of TPP-3310 (see Table 28).

protein heavy chain light chain TPP-5468 SEQ ID NO: 181 SEQ ID NO:182 APP-1574 SEQ ID NO: 183 SEQ ID NO: 184

Table 28: Amino acid sequences of IgG and Fab used for crystal structure determination.

As detailed in Example 1, CEACAM6 domain 1 was expressed from E. coli and refolded. Fab fragments were generated by digesting the antibody with papain, followed by complex formation. Protein crystallography was then used to generate atomic resolution of a single N-terminal domain 1 of human CEACAM6 bound to APP-1574F Fab to define the epitope.

Protein production

A single N-terminal domain 1 of human CEACAM6 (TPP-1794; SEQ-ID 169) was produced as a 6×His fusion protein construct as described in Example 1. The protein was concentrated to 6.7 mg/ml prior to complex formation.

The corresponding Fab fragment of TPP-5468 (human IgG1) was obtained by cleavage with the protease papain. 1 mg of antibody was mixed with 50 μl of immobilized papain (ThermoFisher #20341) in digestion buffer (20 mM sodium phosphate pH 7.0, 10 mM EDTA, 20 mM cysteine-HCl) and incubated at 37°C for 4 hours with continuous stirring. The immobilized papain was removed by centrifugation, and the resulting Fc fragment and uncleaved IgG were removed by passing through MabSelectSURE (GE Healthcare, #11-0034-89 AC). The Fab fragment in the flowthrough was further purified by size exclusion chromatography on a Superdex 75 in 30 mM Tris buffer pH 8.5, 150 mM NaCl and concentrated to 7.2 mg/ml.

For complex formation, purified Fab fragments and human CEACAM6 N-terminal domain 1 were mixed at a ratio of 1 Fab:1.4 human CEACAM6 N-terminal domain 1 for 1 human CEACAM6 N-terminal domain 1 at 4°C for 1 hour. The resulting protein complex was separated by size exclusion chromatography on Superdex 75 in 30 mM Tris buffer, pH 8.5, 150 mM NaCl and further concentrated to 21.2 mg/ml before crystallization.

Crystallization and structure determination

A complex of a single N-terminal domain 1 of human CEACAM6 and the Fab fragment APP-1574 was concentrated to 21.2 mg/ml, centrifuged at 20,000 g for 10 minutes, and selected for crystallization. Crystals for data acquisition were grown by suspended drop vapor diffusion at 20°C. Specifically, 0.2 μl of the complex was mixed with 0.2 μl of a reservoir solution containing 100 mM trisodium citrate, pH 4.9, 19% (w/v) PEG 4000, and 10% (v/v) isopropanol. The droplet was then equilibrated against 80 μl of the same reservoir solution. Prior to data collection, the crystals were rapidly cooled in liquid nitrogen.

Diffraction data were collected at the BESSY II synchrotron source (Helmholtz Zentrum Berlin) using beamline 14-1 and processed using XDS (Kabsch, W. XDS. Acta Cryst. D66, 125-132 (2010). The data for the human CEACAM6 single N-terminal domain 1-Fab fragment APP-1574 complex were resized to 2.7 Å in space group P1, with unit cell dimensions of a = 64.7 Å, b = 65.2 Å, c = 78.6 Å, α = 66.1°, β = 87.2°, and γ = 88.5°. The structure of the complex was solved by molecular replacement using PHASER (McCoy AJ et al., J Appl Cryst (2007). 40, 658-674) using the internal structures of the human CEACAM6 single N-terminal domain 1 and Fab as search models. The final model was constructed using COOT (Emsley, P. et al., ActaCryst D66, 2007). 486-501 (2010)) and improved using CCP4 (Winn, M. D. et al. Acta. Cryst. D67, 235-242 (2011)).

The epitope was defined as residues of the human CEACAM6 single N-terminal domain 1 containing atoms within 5 Å of any atom in the Fab fragment APP-1574, identified using NCONT in the CCP4 program package (Winn, M.D. et al. Acta. Cryst. D67, 235-242 (2011)) and listed in Table 29. Two copies of the human CEACAM6 single N-terminal domain 1-Fab fragment APP-1574 complex exist in the asymmetric unit (the smallest unique unit of the crystal). Only those antibody-contact residues shared by both copies were included as epitope residues.

Epitope

The crystal structure of the human CEACAM6 single N-terminal domain 1-Fab fragment APP-1574 complex was used to identify the epitope of the Fab fragment APP-1574 on CEACAM6. The interaction surface on the human CEACAM6 single N-terminal domain 1 by the Fab fragment APP-1574 is formed by several continuous and discontinuous (i.e., non-continuous) sequences; namely, residues Pro59, Gln60, Asn61, Arg62, Ile63, Gly64, Val83, Ile84, Gly85, Thr86, Gln88, Thr90, Pro91, Ile125, Ser127, Asp128, and Leu129 (numbering according to SEQ-ID: 179; TPP-4639), as detailed in Table 29.

In close direct contact are residues with at least one atom 3.6 Å or less from the antibody. These residues are: Gln60, Asn61, Arg62, Ile63, Val83, Ile84, Gly85, Thr90, Ser127, Asp128, and Leu129 (numbering according to SEQ-ID: 179; TPP-4639).

These residues form an exemplary three-dimensional conformational epitope recognized by the Fab fragment APP-1574 (Figures 9 and 10).

Table 29: Interaction between the single N-terminal domain 1 of human CEACAM6 and Fab APP-1574.

CEACAM6 N-terminal domain 1 residues are numbered as in SEQ ID NO: 169. Antibody residues are numbered based on their linear amino acid sequences (SEQ ID NO: 183 and SEQ ID NO: 184) and labeled with the corresponding chain ("H" for heavy chain, "L" for light chain). A single N-terminal domain 1 residue of human CEACAM6 is shown here with at least one atom within 5 Å of any atom in the Fab fragment APP-1574 to account for potential water-mediated interactions.

Upon careful analysis of the epitope, it became apparent that isoleucine-63 of human CEACAM6 (according to SEQ-ID NO: 179) is the central part of the epitope. The isoleucine side chain has good shape complementarity with APP-1574. Modeling showed that a leucine at this position in cynomolgus monkey CEACAM6 can be accommodated sterically without disrupting the interaction. This explains the retained binding activity for cynomolgus monkey CEACAM6 activity and underlies the human-cynomolgus monkey cross-reactivity. In contrast, a phenylalanine at this position (as in human CEACAM1, human CEACAM3, and human CEACAM5) cannot be accommodated sterically and would result in a loss of binding activity. This underlies the selectivity of CEACAM6.

Thus, recognition of Isoleucine-63 (according to SEQ-ID NO: 179) represents the most important "selectivity modulator" between target CEACAMs. The APP-1574 recognition pattern for human CEACAM6 optimally exploits key residue differences between target and non-target CEACAM6. Previous structural analysis of the Fab fragment of TPP-1679 (whose selectivity profile is inadequate, see Example 4) in complex with N-terminal domain 1 also identified Isoleucine-63 (according to SEQ-ID NO: 179) as a potential selectivity switch (data not shown). However, since the molecular recognition mechanism of TPP-1679 is distinct, and thus, Isoleucine-63 (according to SEQ-ID NO: 179) is located peripherally to the binding site, it has been difficult to exploit.

In summary, binders that optimally exploit the selectivity and cross-reactivity determining residue isoleucine-63 (according to SEQ-ID 0: 179) for binding to CEACAM6 in addition to the other residues that form the epitope (Table 29), and that also allow accommodation of a leucine at this position instead of a phenylalanine, will be CEACAM6 selective, but at the same time human-cynomolgus CEACAM6 cross-reactive.

mutagenesis

To confirm the findings and predictions from the structural analysis in the binding studies, the following mutants of the N-terminal domain 1 of human CEACAM6 were generated:

Table 30: Amino acid sequences of proteins used in mutation binding studies.

Protein was expressed in Escherichia coli, refolded and purified as described in Example 1. The comparative binding activity with domain 1 wild-type protein and two single mutations was determined by ELISA. 1.5 ug/ml protein solution in PBS was coated onto 384 Nunc MaxiSorp plates (Sigma, P6491) overnight. The plates were washed with PBS/T and blocked with Smart block (CANDOR Bioscience GmbH, 113125). Subsequently, a dilution series of TPP-3310, TPP-1679 and isotype control antibodies was applied to the wells. After washing with PBS/T, the bound antibodies were detected with anti-human IgG Fc POD (Sigma, A0170) and 10 μM Amplex Red solutions (Thermo, A12222). Positive binding signals were detected via fluorescence (excitation 535 nm/emission 590 nm). Table 32 shows the binding activity of TPP-3310 to the wild-type domain 1 protein and the mutation of isoleucine 63 (as in SEQ-ID NO: 179) to leucine (as in cynomolgus monkey). Mutation of amino acid position 63 (as in SEQ-ID NO: 179) to phenylalanine (as in human CEACAM1, human CEACAM3, and human CEACAM5) resulted in a complete loss of binding. As a control to demonstrate efficient refolding of the CEACAM6 N-terminal domain 1 protein, TPP-1679 (see Example 4) was used, for which binding was observed to a similar extent to all antigens tested in Table 31.

Antibody Human CEACAM6-Domain 1-His Wild-type TPP-1794; SEQ-ID NO:169 Human CEACAM6-Domain 1-His　　　I30L(I63L, according to SEQ-ID NO:179)TPP-8697; SEQ-ID NO:185 Human CEACAM6-Domain 1-His　　　I30F(I63F, according to SEQ-ID NO:179)TPP-8698; SEQ-ID NO:186 TPP-3310 + + - TPP-1679 + + + Isotype control - - -

"+" indicates that binding was detected; "-" indicates that no binding was detected

Table 31: Binding activity to CEACAM6 N-terminal domain 1 mutants.

In summary, in the context of human CEACAM6 N-terminal domain 1, the authentic human-cynomolgus CEACAM6 cross-reactive antibody TPP-3310 (which is also selective with respect to other human paralogs) can tolerate the I63L substitution (according to SEQ-ID: 179) (corresponding to the cynomolgus CEACAM6 residue), but not the I63F substitution (according to SEQ-ID: 179) (the corresponding residue in the human paralogs CEACAM1, CEACAM3 and CEACAM5), which is consistent with the results obtained from X-ray crystallography and confirms our predictions.

Example 17: Analysis of T cell-mediated cytotoxicity in the presence of CEACAM6 antibodies

The effects of anti-CEACAM6 antibodies on T cell-mediated cytotoxicity were investigated in cytotoxicity experiments using co-cultures of CEACAM6-positive tumor cells and T cells derived from various sources. These T cells were either CD8 ⁺ survivin T cells from pancreatic cancer or patient-derived T cells. For these tumor cell killing experiments, an impedance-based cytotoxicity assay (xCELLigence) system was used.

Survivin-peptide-specific CD8 ⁺ T cell clones were generated and expanded in vitro as described in Example 11. Pancreatic cancer tumor-infiltrating lymphocyte cell lines (TILs) were isolated from fresh primary cultures of tumor tissue from surgical procedures. Briefly, fresh primary tissue material was cut into small pieces and cultured in X-Vivo-15 medium (Lonza) containing 2% human serum albumin, 2.5 μg/ml amphotericin, 20 μg/ml gentamicin, 1% penicillin/streptomycin, and 6000 IU/IL-2 in small dishes for 10-18 days. Thereafter, cells were harvested from the supernatant and frozen or used directly in the "Rapid Expansion Protocol" (REP). For rapid expansion of TILs, frozen TILs were gently thawed and cultured at 0.6*10 6 cells/ml in complete lymphocyte culture medium CLM RPMI-1640 (Life Technologies #21875034), 10% human AB serum (MILAN Analytica #000083), 1% penicillin/streptomycin (Life Technologies #15140122), 1% ml HEPES (Life Technolgies #15630056), 0.01% β-mercaptoethanol [stock ⁵⁰ mM] (Life Technologies #31350010)) with 6000 IU/ml IL-2 for 1 day. TILs were harvested and expanded in 400 ml of REP medium (50% CLM mixed with 50% AIM-V serum-free medium (Gibco #12055091) containing 3000 IU/ml IL-2 and 30 ng/ml OKT-3 antibody (eBioscience #16-0037-85) in G-REX-100 Flasks (WilsonWolf #80500S) with 60 Gy irradiated feeder PBMCs from three different donors at a 1:100 ratio. The cells were cultured and split as described in Jin et al., J Immunother. 2012 Apr;35(3):283-92. After 14 days, the cells were harvested and frozen in aliquots. Prior to the co-culture cytotoxicity assay, separate aliquots of TIL were gently thawed and cultured with 0.6*10 ⁶ cells/ml in CLM containing 6000 IU/ml IL-2 for 2 days and in CLM without IL-2 for 1 day.

Tumor cells were cultured according to standard protocols and the provider's instructions.

T cell-mediated cytotoxicity is analyzed in an impedance-based cytotoxicity assay (xCELLigence) system. In this label-free assay system, cytotoxicity is directly and continuously measured over a long period of about 100-150 hours (real time). Adherent tumor cells are attached to the microelectrodes at the bottom of a 96-well E plate (E-Plate VIEW 96 PET; ACEA Biosciences #ID: H000568), which changes the electrical impedance of these electrodes. This is monitored as an increase in a dimensionless "cell index". After tumor cell adhesion (~24 hours), antibodies and T cells are added to the wells. If the T cells exert cytotoxic activity, they cause tumor cell lysis and detach from the electrodes. This detachment changes the impedance of the wells and is measured as a decrease in "cell index" or "normalized cell index", which is a "cell index" normalized at the time point added for T cells. Individual T cells do not affect the electrical impedance of the electrode, so only the cell lysis of tumor cells is measured. (Peper et al., J Immunol Methods. 2014 Mar;405:192-8).

In the first experiment, we determined that the tumor cell killing observed in this assay system was T cell dose-dependent and functioned for different tumor cell:T cell ratios and different T cell sources (survivin-peptide-specific CD8 ⁺ T cells, TILs from pancreatic cancer patients).

We then investigated the effect of anti-CEACAM6 antibodies on the cytolytic potency of survivin T cells. CEACAM6-positive breast cancer KS or CEACAM6-transfected colon cancer HCT-116 (HCT116-hC6) cells were added to 96-well plates for 24 hours, followed by the addition of survivin-peptide-specific T cells at varying cell ratios along with anti-CEACAM6 mAbs. The co-culture period was followed for ~100 hours. In these experiments, we observed improved T-cell-dependent cytotoxicity in the presence of ~21% of the anti-CEACAM6 antibodies TPP-3310 and TPP-3470 on both cell lines. The results are shown exemplarily for one cell ratio in Figures 11A and B. Notably, even survivin-peptide-specific CD8 ⁺ T cells alone exhibited a high cytotoxic effect of 45-62%, which is most likely due to pre-activation of the cultured T cells and is therefore considered background cytolysis. Overall, the increase in IFN-γ secretion observed in the previous ELISA assay translated into a cytotoxic effect within approximately 24 hours of co-culture. We conclude that treatment of CEACAM6-positive tumor cells with anti-CEACAM6 antibody resulted in improved survivin-peptide-specific CD8 ⁺ T cell-mediated killing of both tumor cell lines.

In subsequent experiments, we tested the effect of CEACAM6 antibodies on the cytolytic activity of TIL cells derived from patients with pancreatic cancer. Thus, the CEACAM6-positive lung cancer cell line HCC2935 was added to 96-well plates and cultured for 24 hours. TILs were then added at varying ratios in the presence of CEACAM6 antibodies (30 μg/ml) and the bispecific antibody anti-CD3 x anti-EPCAM IgG (0.25 ng/ml) (Marmé et al., Int J Cancer. 2002 Sep 10;101(2):183-9; Salnikov et al., J Cell Mol Med. 2009 Sep;13(9B):4023-33) to allow HLA-independent T cell-mediated tumor cell killing. In the presence of the anti-CEACAM6 antibodies TPP-3310 and TPP-3470, we observed a complete decrease in impedance that was not observed in the presence of isotype-matched control antibodies. The decrease in impedance was interpreted as complete cytolytic killing of the target cell line HCC2935. In additional experiments, it was demonstrated that the effect of the CEACAM6 antibody TPP-3310 was dose-dependent, and _IC50 values of 0.62-0.21 μg/ml were determined. Figure 12 exemplifies the results for TIL-12.

In summary, these experiments show that the CEACAM6 antibodies of the present invention have the potential to effectively block the immunosuppressive receptor CEACAM6 and improve the cytotoxic efficacy of not only model T cells but also patient-derived tumor-infiltrating lymphocytes against CEACAM6-positive tumor cells.

Sequence Listing

Table 32: Correlation of SEQ ID NOs to TPP-IDs and related sequence features (heavy and light chains, variable regions, complementarity determining regions (CDRs) of antibodies) for proteins (PRTs) and nucleic acids (DNAs).

Sequence Listing

<110> Bayer Pharma AG

<120> Anti-CEACAM6 antibodies and their uses

<130> BHC 14 1 064

<160> 186

<170> PatentIn Version 3.5

<210> 1

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-1173, h16C3-hIgG1, heavy chain

<400> 1

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Leu Ile Ser Thr Tyr Ser Gly Asp Thr Lys Tyr Asn Gln Asn Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Asp Tyr Ser Gly Ser Arg Tyr Trp Phe Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly

450

<210> 2

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-1173, h16C3-hIgG1, light chain

<400> 2

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Asn Ile Tyr Gly Ala

20 25 30

Leu Asn Trp Tyr Gln Arg Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Asn Leu Ala Thr Gly Met Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Val Leu Ser Ser Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 3

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-2971, 792.15H12C9, VH

<400> 3

Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Tyr

20 25 30

Gly Ile Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Asp Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Asn Thr Ala

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Asn Asn Gln Val

65 70 75 80

Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Ser Leu Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 4

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-2971, 792.15H12C9, HCDR1

<400> 4

Thr Tyr Gly Ile Gly Val Gly

1 5

<210> 5

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-2971, 792.15H12C9, HCDR2

<400> 5

His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Asn Thr Ala Leu Lys Ser

1 5 10 15

<210> 6

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-2971, 792.15H12C9, HCDR3

<400> 6

Ile Ser Leu Pro Tyr Phe Asp Tyr

1 5

<210> 7

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-2971, 792.15H12C9, VL

<400> 7

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Met Gln Ser

65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 8

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-2971, 792.15H12C9, LCDR1

<400> 8

Lys Ala Ser Gln Asn Val Gly Thr Ala Val Ala

1 5 10

<210> 9

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-2971, 792.15H12C9, LCDR2

<400> 9

Ser Ala Ser Asn Arg Tyr Thr

1 5

<210> 10

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-2971, 792.15H12C9, LCDR3

<400> 10

Gln Gln Tyr Ser Ser Tyr Pro Leu Thr

1 5

<210> 11

<211> 129

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-2971, 792.15H12C9, heavy chain

<400> 11

Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Tyr

20 25 30

Gly Ile Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Asp Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Asn Thr Ala

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Asn Asn Gln Val

65 70 75 80

Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Ser Leu Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro

115 120 125

Leu

<210> 12

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-2971, 792.15H12C9, light chain

<400> 12

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Met Gln Ser

65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala

100 105 110

Pro Thr Val Ser Ile Phe Pro Pro Ser Ser

115 120

<210> 13

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3186, 792.11G2D10, VH

<400> 13

Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Tyr

20 25 30

Gly Ile Gly Val Gly Cys Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Asn Thr Ala

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Asn Asn Gln Val

65 70 75 80

Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Ser Leu Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 14

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3186, 792.11G2D10, HCDR1

<400> 14

Thr Tyr Gly Ile Gly Val Gly

1 5

<210> 15

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3186, 792.11G2D10, HCDR2

<400> 15

His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Asn Thr Ala Leu Lys Ser

1 5 10 15

<210> 16

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3186, 792.11G2D10, HCDR3

<400> 16

Ile Ser Leu Pro Tyr Phe Asp Tyr

1 5

<210> 17

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3186, 792.11G2D10, VL

<400> 17

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Asn Met Gln Ser

65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 18

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3186, 792.11G2D10, LCDR1

<400> 18

Lys Ala Ser Gln Asn Val Gly Thr Ala Val Ala

1 5 10

<210> 19

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3186, 792.11G2D10, LCDR2

<400> 19

Ser Ala Ser Asn Arg Tyr Thr

1 5

<210> 20

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3186, 792.11G2D10, LCDR3

<400> 20

Gln Gln Tyr Ser Ser Tyr Pro Leu Thr

1 5

<210> 21

<211> 129

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3186, 792.11G2D10, heavy chain

<400> 21

Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Tyr

20 25 30

Gly Ile Gly Val Gly Cys Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Asn Thr Ala

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Asn Asn Gln Val

65 70 75 80

Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Ser Leu Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro

115 120 125

Leu

<210> 22

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3186, 792.11G2D10, light chain

<400> 22

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Asn Met Gln Ser

65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala

100 105 110

Pro Thr Val Ser Ile Phe Pro Pro Ser Ser

115 120

<210> 23

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3187, 792.15C4F4, VH

<400> 23

Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Thr Thr Tyr

20 25 30

Gly Ile Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Asn Thr Ala

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Asn Asn Gln Val

65 70 75 80

Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Ser Leu Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 24

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3187, 792.15C4F4, HCDR1

<400> 24

Thr Tyr Gly Ile Gly Val Gly

1 5

<210> 25

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3187, 792.15C4F4, HCDR2

<400> 25

His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Asn Thr Ala Leu Lys Ser

1 5 10 15

<210> 26

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3187, 792.15C4F4, HCDR3

<400> 26

Ile Ser Leu Pro Tyr Phe Asp Tyr

1 5

<210> 27

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3187, 792.15C4F4, VL

<400> 27

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Met Gln Ser

65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Asn Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 28

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3187, 792.15C4F4, LCDR1

<400> 28

Lys Ala Ser Gln Asn Val Gly Thr Ala Val Ala

1 5 10

<210> 29

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3187, 792.15C4F4, LCDR2

<400> 29

Ser Ala Ser Asn Arg Tyr Thr

1 5

<210> 30

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3187, 792.15C4F4, LCDR3

<400> 30

Gln Gln Tyr Asn Asn Tyr Pro Leu Thr

1 5

<210> 31

<211> 129

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3187, 792.15C4F4, heavy chain

<400> 31

Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Thr Thr Tyr

20 25 30

Gly Ile Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Asn Thr Ala

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Asn Asn Gln Val

65 70 75 80

Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Ser Leu Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro

115 120 125

Leu

<210> 32

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3187, 792.15C4F4, light chain

<400> 32

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Met Gln Ser

65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Asn Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala

100 105 110

Pro Thr Val Ser Ile Phe Pro Pro Ser Ser

115 120

<210> 33

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3308, TPP-2971X1-hIgG2Kappa, VH

<400> 33

Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Tyr

20 25 30

Gly Ile Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Asp Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Asn Thr Ala

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Asn Asn Gln Val

65 70 75 80

Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Ser Leu Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 34

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3308, TPP-2971X1-hIgG2Kappa, HCDR1

<400> 34

Thr Tyr Gly Ile Gly Val Gly

1 5

<210> 35

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3308, TPP-2971X1-hIgG2Kappa, HCDR2

<400> 35

His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Asn Thr Ala Leu Lys Ser

1 5 10 15

<210> 36

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3308, TPP-2971X1-hIgG2Kappa, HCDR3

<400> 36

Ile Ser Leu Pro Tyr Phe Asp Tyr

1 5

<210> 37

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3308, TPP-2971X1-hIgG2Kappa, VL

<400> 37

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Met Gln Ser

65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 38

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3308, TPP-2971X1-hIgG2Kappa, LCDR1

<400> 38

Lys Ala Ser Gln Asn Val Gly Thr Ala Val Ala

1 5 10

<210> 39

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3308, TPP-2971X1-hIgG2Kappa, LCDR2

<400> 39

Ser Ala Ser Asn Arg Tyr Thr

1 5

<210> 40

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3308, TPP-2971X1-hIgG2Kappa, LCDR3

<400> 40

Gln Gln Tyr Ser Ser Tyr Pro Leu Thr

1 5

<210> 41

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3308, TPP-2971X1-hIgG2Kappa, VH

<400> 41

caggtcacac tgaaagagag cggccctggc atcctgcagc ccagccagac cctgagcctg 60

acctgcagct tcagcggctt cagcctgagc acctacggca tcggcgtggg ctggatcaga 120

cagcccagcg gcaaggacct ggaatggctg gcccacatct ggtggaacga caacaagtac 180

tacaacaccg ccctgaagtc ccggctgacc atcagcaagg acaccagcaa caaccaggtg 240

ttcctgaaga tcgccagcgt ggacaccgcc gataccgcca cctactactg cgcccggatc 300

agcctgccct acttcgacta ctggggccag ggcaccaccc tgaccgtgtc ctca 354

<210> 42

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3308, TPP-2971X1-hIgG2Kappa, VL

<400> 42

gacatcgtga tgacccag ccagaaattc atgagcacca gcgtgggcga ccgggtgtcc 60

atcacatgca aggccagcca gaacgtgggc accgccgtgg cctggtatca gcagaagccc 120

ggccagagcc ccaagctgct gatctacagc gccagcaacc ggtacaccgg cgtgcccgac 180

agattcacag gcagcggcag cggcaccgac ttcaccctga ccatcagcaa catgcagagc 240

gaggacctgg ccgactactt ctgccagcag tacagcagct accccctgac cttcggagcc 300

ggcaccaagc tggaactgaa a 321

<210> 43

<211> 443

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3308, TPP-2971X1-hIgG2Kappa, heavy chain

<400> 43

Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Tyr

20 25 30

Gly Ile Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Asp Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Asn Thr Ala

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Asn Asn Gln Val

65 70 75 80

Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Ser Leu Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys

210 215 220

Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr

290 295 300

Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440

<210> 44

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3308, TPP-2971X1-hIgG2Kappa, light chain

<400> 44

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Met Gln Ser

65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 45

<211> 1329

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3308, TPP-2971X1-hIgG2Kappa, heavy chain

<400> 45

caggtcacac tgaaagagag cggccctggc atcctgcagc ccagccagac cctgagcctg 60

acctgcagct tcagcggctt cagcctgagc acctacggca tcggcgtggg ctggatcaga 120

cagcccagcg gcaaggacct ggaatggctg gcccacatct ggtggaacga caacaagtac 180

tacaacaccg ccctgaagtc ccggctgacc atcagcaagg acaccagcaa caaccaggtg 240

ttcctgaaga tcgccagcgt ggacaccgcc gataccgcca cctactactg cgcccggatc 300

agcctgccct acttcgacta ctggggccag ggcaccaccc tgaccgtgtc ctcagccagc 360

accaagggcc ccagcgtgtt ccctctggcc ccttgtagca gaagcaccag cgagtctaca 420

gccgccctgg gctgcctcgt gaaggactac tttcccgagc ccgtgaccgt gtcctggaac 480

tctggcgctc tgacaagcgg cgtgcacacc tttccagccg tgctgcagag cagcggcctg 540

tactctctga gcagcgtcgt gacagtgccc agcagcaact tcggcaccca gacctacacc 600

tgtaacgtgg accacaagcc cagcaacacc aaggtggaca agaccgtgga acggaagtgc 660

tgcgtggaat gccccccttg tcctgcccct ccagtggctg gcccttccgt gttcctgttc 720

cccccaaagc ccaaggacac cctgatgatc agccggaccc ccgaagtgac ctgcgtggtg 780

gtggatgtgt cccacgagga ccccgaggtg cagttcaatt ggtacgtgga cggcgtggaa 840

gtgcacaacg ccaagaccaa gcccagagag gaacagttca acagcacctt ccgggtggtg 900

tccgtgctga ccgtggtgca tcaggactgg ctgaacggca aagagtacaa gtgcaaggtg 960

tccaacaagg gcctgcctgc ccccatcgag aaaaccatca gcaagaccaa aggccagccc 1020

cgcgagcccc aggtgtacac actgcctcca agccgggaag agatgaccaa gaaccaggtg 1080

tccctgacct gtctcgtgaa aggcttctac ccctccgata tcgccgtgga atgggagagc 1140

aacggccagc ccgagaacaa ctacaagacc accccccccca tgctggacag cgacggctca 1200

ttcttcctgt acagcaagct gacagtggac aagtcccggt ggcagcaggg caacgtgttc 1260

agctgcagcg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgagcctg 1320

agccctggc 1329

<210> 46

<211> 642

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3308, TPP-2971X1-hIgG2Kappa, light chain

<400> 46

gacatcgtga tgacccag ccagaaattc atgagcacca gcgtgggcga ccgggtgtcc 60

atcacatgca aggccagcca gaacgtgggc accgccgtgg cctggtatca gcagaagccc 120

ggccagagcc ccaagctgct gatctacagc gccagcaacc ggtacaccgg cgtgcccgac 180

agattcacag gcagcggcag cggcaccgac ttcaccctga ccatcagcaa catgcagagc 240

gaggacctgg ccgactactt ctgccagcag tacagcagct accccctgac cttcggagcc 300

ggcaccaagc tggaactgaa acgaaccgtg gccgctccca gcgtgttcat cttcccacct 360

agcgacgagc agctgaagtc cggcacagcc tctgtcgtgt gcctgctgaa caacttctac 420

ccccgcgagg ccaaggtgca gtggaaggtg gacaatgccc tgcagagcgg caacagccag 480

gaaagcgtga ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540

ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600

ctgtctagcc ccgtgaccaa gagcttcaac cggggcgagt gt 642

<210> 47

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3310, TPP-2971HU1-hIgG2Kappa, VH

<400> 47

Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Tyr

20 25 30

Gly Ile Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Ser Thr Ser

50 55 60

Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Ser Leu Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 48

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3310, TPP-2971HU1-hIgG2Kappa, HCDR1

<400> 48

Thr Tyr Gly Ile Gly Val Gly

1 5

<210> 49

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3310, TPP-2971HU1-hIgG2Kappa, HCDR2

<400> 49

His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Ser Thr Ser Leu Lys Thr

1 5 10 15

<210> 50

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3310, TPP-2971HU1-hIgG2Kappa, HCDR3

<400> 50

Ile Ser Leu Pro Tyr Phe Asp Tyr

1 5

<210> 51

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3310, TPP-2971HU1-hIgG2Kappa, VL

<400> 51

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 52

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3310, TPP-2971HU1-hIgG2Kappa, LCDR1

<400> 52

Lys Ala Ser Gln Asn Val Gly Thr Ala Val Ala

1 5 10

<210> 53

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3310, TPP-2971HU1-hIgG2Kappa, LCDR2

<400> 53

Ser Ala Ser Asn Arg Tyr Thr

1 5

<210> 54

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3310, TPP-2971HU1-hIgG2Kappa, LCDR3

<400> 54

Gln Gln Tyr Ser Ser Tyr Pro Leu Thr

1 5

<210> 55

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3310, TPP-2971HU1-hIgG2Kappa, VH

<400> 55

caagtgaccc tgagagagtc cggccctgcc ctcgtgaagc ctacccagac cctgacactg 60

acctgcacct tctccggctt ctccctgtcc acctacggca tcggcgtggg ctggatcaga 120

cagcctcctg gcaaggccct ggaatggctg gctcacatct ggtggaacga caacaagtac 180

tactccacct ccctgaaaac ccggctgacc atctccaagg acacctccaa gaaccaggtg 240

gtgctgacca tgaccaacat ggaccccgtg gacaccgcca cctactactg cgccagaatc 300

tccctgccct acttcgacta ctggggccag ggcaccacac tgaccgtcag ctca 354

<210> 56

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3310, TPP-2971HU1-hIgG2Kappa, VL

<400> 56

gatatccagc tgacccagtc ccccagcttc ctgtctgcct ctgtgggcga cagagtgacc 60

atcacatgca aggcctccca gaacgtgggc accgccgtgg cttggtatca gcagaagcct 120

ggcaaggccc ccaagctgct gatctactcc gcctccaacc ggtacaccgg cgtgccctct 180

agattctccg gctctggctc tggcaccgag tttaccctga ccatctccag cctgcagccc 240

gaggacttcg ccacctacta ctgccagcag tactcctcct accccctgac ctttggcgga 300

ggcaccaagg tggaaatcaa g 321

<210> 57

<211> 443

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3310, TPP-2971HU1-hIgG2Kappa, heavy chain

<400> 57

Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Tyr

20 25 30

Gly Ile Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Ser Thr Ser

50 55 60

Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Ser Leu Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys

210 215 220

Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr

290 295 300

Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440

<210> 58

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3310, TPP-2971HU1-hIgG2Kappa, light chain

<400> 58

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 59

<211> 1329

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3310, TPP-2971HU1-hIgG2Kappa, heavy chain

<400> 59

caagtgaccc tgagagagtc cggccctgcc ctcgtgaagc ctacccagac cctgacactg 60

acctgcacct tctccggctt ctccctgtcc acctacggca tcggcgtggg ctggatcaga 120

cagcctcctg gcaaggccct ggaatggctg gctcacatct ggtggaacga caacaagtac 180

tactccacct ccctgaaaac ccggctgacc atctccaagg acacctccaa gaaccaggtg 240

gtgctgacca tgaccaacat ggaccccgtg gacaccgcca cctactactg cgccagaatc 300

tccctgccct acttcgacta ctggggccag ggcaccacac tgaccgtcag ctcagcttcc 360

accaagggcc cctccgtgtt ccctctggcc ccttgctccc ggtccacctc tgagtctacc 420

gccgctctgg gctgcctggt gaaagactac ttccccgagc ccgtgaccgt gtcctggaac 480

tctggcgccc tgacctccgg cgtgcacacc tttccagccg tgctgcagtc ctccggcctg 540

tactccctgt cctccgtggt gacagtgccc tcctccaact tcggcaccca gacctacacc 600

tgtaacgtgg accacaagcc ctccaacacc aaggtggaca agaccgtgga acggaagtgc 660

tgcgtggaat gcccaccctg tcctgctcca cctgtggctg gccccagcgt gttcctgttc 720

cccccaaagc ccaaggacac cctgatgatc tcccggaccc ccgaagtgac ctgcgtggtg 780

gtggacgtgt cccacgagga ccccgaggtg cagttcaatt ggtacgtgga cggcgtggaa 840

gtgcacaacg ccaagaccaa gcccagagag gaacagttca actccacctt ccgggtggtg 900

tccgtgctga ccgtggtgca tcaggactgg ctgaacggca aagagtacaa gtgcaaggtc 960

tccaacaagg gcctgcctgc ccccatcgaa aagaccatca gcaagaccaa gggccagccc 1020

cgcgagcccc aggtgtacac actgcccccc agccgggaag agatgaccaa gaaccaggtg 1080

tccctgacct gtctggtgaa aggcttctac ccctccgaca ttgccgtgga atgggagtcc 1140

aacggacagc ctgagaacaa ctacaagacc accccccccca tgctggactc cgacggctca 1200

ttcttcctgt actccaagct gacagtggac aagtcccggt ggcagcaggg caacgtgttc 1260

tcctgctccg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgtccctg 1320

agccccggc 1329

<210> 60

<211> 642

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3310, TPP-2971HU1-hIgG2Kappa, light chain

<400> 60

gatatccagc tgacccagtc ccccagcttc ctgtctgcct ctgtgggcga cagagtgacc 60

atcacatgca aggcctccca gaacgtgggc accgccgtgg cttggtatca gcagaagcct 120

ggcaaggccc ccaagctgct gatctactcc gcctccaacc ggtacaccgg cgtgccctct 180

agattctccg gctctggctc tggcaccgag tttaccctga ccatctccag cctgcagccc 240

gaggacttcg ccacctacta ctgccagcag tactcctcct accccctgac ctttggcgga 300

ggcaccaagg tggaaatcaa gcggaccgtg gccgctccct ccgtgtttat cttcccaccc 360

tccgacgagc agctgaagtc cggcacagct tccgtcgtgt gcctgctgaa caacttctac 420

ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caactcccag 480

gaatccgtga ccgagcagga ctccaaggac agcacctact ccctgtcctc caccctgacc 540

ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600

ctgtctagcc ccgtgaccaa gtctttcaac cggggcgagt gt 642

<210> 61

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3322, TPP-3186X1-hIgG2, VH

<400> 61

Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Tyr

20 25 30

Gly Ile Gly Val Gly Cys Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Asn Thr Ala

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Asn Asn Gln Val

65 70 75 80

Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Ser Leu Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 62

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3322, TPP-3186X1-hIgG2, HCDR1

<400> 62

Thr Tyr Gly Ile Gly Val Gly

1 5

<210> 63

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3322, TPP-3186X1-hIgG2, HCDR2

<400> 63

His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Asn Thr Ala Leu Lys Ser

1 5 10 15

<210> 64

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3322, TPP-3186X1-hIgG2, HCDR3

<400> 64

Ile Ser Leu Pro Tyr Phe Asp Tyr

1 5

<210> 65

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3322, TPP-3186X1-hIgG2, VL

<400> 65

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Asn Met Gln Ser

65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 66

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3322, TPP-3186X1-hIgG2, LCDR1

<400> 66

Lys Ala Ser Gln Asn Val Gly Thr Ala Val Ala

1 5 10

<210> 67

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3322, TPP-3186X1-hIgG2, LCDR2

<400> 67

Ser Ala Ser Asn Arg Tyr Thr

1 5

<210> 68

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3322, TPP-3186X1-hIgG2, LCDR3

<400> 68

Gln Gln Tyr Ser Ser Tyr Pro Leu Thr

1 5

<210> 69

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3322, TPP-3186X1-hIgG2, VH

<400> 69

caggtcacac tgaaagagag cggccctggc atcctgcagc ccagccagac cctgagcctg 60

acctgcagct tcagcggctt cagcctgagc acctacggca tcggcgtggg ctgcatcaga 120

cagcccagcg gcaagggcct ggaatggctg gcccacatct ggtggaacga caacaagtac 180

tacaacaccg ccctgaagtc ccggctgacc atcagcaagg acaccagcaa caaccaggtg 240

ttcctgaaga tcgccagcgt ggacaccgcc gataccgcca cctactactg cgcccggatc 300

agcctgccct acttcgacta ctggggccag ggcaccaccc tgaccgtgtc ctca 354

<210> 70

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3322, TPP-3186X1-hIgG2, VL

<400> 70

gacatcgtga tgacccag ccagaaattc atgagcacca gcgtgggcga ccgggtgtcc 60

atcacatgca aggccagcca gaacgtgggc accgccgtgg cctggtatca gcagaagccc 120

ggccagagcc ccaagctgct gatctacagc gccagcaacc ggtacaccgg cgtgcccgac 180

agattcacag gcagcggcag cggcaccgac ttcaccttca ccatcagcaa catgcagagc 240

gaggacctgg ccgactactt ctgccagcag tacagcagct accccctgac cttcggagcc 300

ggcaccaagc tggaactgaa a 321

<210> 71

<211> 443

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3322, TPP-3186X1-hIgG2, heavy chain

<400> 71

Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Tyr

20 25 30

Gly Ile Gly Val Gly Cys Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Asn Thr Ala

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Asn Asn Gln Val

65 70 75 80

Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Ser Leu Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys

210 215 220

Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr

290 295 300

Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440

<210> 72

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3322, TPP-3186X1-hIgG2, light chain

<400> 72

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Asn Met Gln Ser

65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 73

<211> 1329

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3322, TPP-3186X1-hIgG2, heavy chain

<400> 73

caggtcacac tgaaagagag cggccctggc atcctgcagc ccagccagac cctgagcctg 60

acctgcagct tcagcggctt cagcctgagc acctacggca tcggcgtggg ctgcatcaga 120

cagcccagcg gcaagggcct ggaatggctg gcccacatct ggtggaacga caacaagtac 180

tacaacaccg ccctgaagtc ccggctgacc atcagcaagg acaccagcaa caaccaggtg 240

ttcctgaaga tcgccagcgt ggacaccgcc gataccgcca cctactactg cgcccggatc 300

agcctgccct acttcgacta ctggggccag ggcaccaccc tgaccgtgtc ctcagccagc 360

accaagggcc ccagcgtgtt ccctctggcc ccttgtagca gaagcaccag cgagtctaca 420

gccgccctgg gctgcctcgt gaaggactac tttcccgagc ccgtgaccgt gtcctggaac 480

tctggcgctc tgacaagcgg cgtgcacacc tttccagccg tgctgcagag cagcggcctg 540

tactctctga gcagcgtcgt gacagtgccc agcagcaact tcggcaccca gacctacacc 600

tgtaacgtgg accacaagcc cagcaacacc aaggtggaca agaccgtgga acggaagtgc 660

tgcgtggaat gccccccttg tcctgcccct ccagtggctg gcccttccgt gttcctgttc 720

cccccaaagc ccaaggacac cctgatgatc agccggaccc ccgaagtgac ctgcgtggtg 780

gtggatgtgt cccacgagga ccccgaggtg cagttcaatt ggtacgtgga cggcgtggaa 840

gtgcacaacg ccaagaccaa gcccagagag gaacagttca acagcacctt ccgggtggtg 900

tccgtgctga ccgtggtgca tcaggactgg ctgaacggca aagagtacaa gtgcaaggtg 960

tccaacaagg gcctgcctgc ccccatcgag aaaaccatca gcaagaccaa aggccagccc 1020

cgcgagcccc aggtgtacac actgcctcca agccgggaag agatgaccaa gaaccaggtg 1080

tccctgacct gtctcgtgaa aggcttctac ccctccgata tcgccgtgga atgggagagc 1140

aacggccagc ccgagaacaa ctacaagacc accccccccca tgctggacag cgacggctca 1200

ttcttcctgt acagcaagct gacagtggac aagtcccggt ggcagcaggg caacgtgttc 1260

agctgcagcg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgagcctg 1320

agccctggc 1329

<210> 74

<211> 642

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3322, TPP-3186X1-hIgG2, light chain

<400> 74

gacatcgtga tgacccag ccagaaattc atgagcacca gcgtgggcga ccgggtgtcc 60

atcacatgca aggccagcca gaacgtgggc accgccgtgg cctggtatca gcagaagccc 120

ggccagagcc ccaagctgct gatctacagc gccagcaacc ggtacaccgg cgtgcccgac 180

agattcacag gcagcggcag cggcaccgac ttcaccttca ccatcagcaa catgcagagc 240

gaggacctgg ccgactactt ctgccagcag tacagcagct accccctgac cttcggagcc 300

ggcaccaagc tggaactgaa acgaaccgtg gccgctccca gcgtgttcat cttcccacct 360

agcgacgagc agctgaagtc cggcacagcc tctgtcgtgt gcctgctgaa caacttctac 420

ccccgcgagg ccaaggtgca gtggaaggtg gacaatgccc tgcagagcgg caacagccag 480

gaaagcgtga ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540

ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600

ctgtctagcc ccgtgaccaa gagcttcaac cggggcgagt gt 642

<210> 75

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3323, TPP-3187X1-hIgG2, VH

<400> 75

Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Thr Thr Tyr

20 25 30

Gly Ile Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Asn Thr Ala

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Asn Asn Gln Val

65 70 75 80

Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Ser Leu Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 76

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3323, TPP-3187X1-hIgG2, HCDR1

<400> 76

Thr Tyr Gly Ile Gly Val Gly

1 5

<210> 77

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3323, TPP-3187X1-hIgG2, HCDR2

<400> 77

His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Asn Thr Ala Leu Lys Ser

1 5 10 15

<210> 78

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3323, TPP-3187X1-hIgG2, HCDR3

<400> 78

Ile Ser Leu Pro Tyr Phe Asp Tyr

1 5

<210> 79

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3323, TPP-3187X1-hIgG2, VL

<400> 79

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Met Gln Ser

65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Asn Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 80

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3323, TPP-3187X1-hIgG2, LCDR1

<400> 80

Lys Ala Ser Gln Asn Val Gly Thr Ala Val Ala

1 5 10

<210> 81

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3323, TPP-3187X1-hIgG2, LCDR2

<400> 81

Ser Ala Ser Asn Arg Tyr Thr

1 5

<210> 82

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3323, TPP-3187X1-hIgG2, LCDR3

<400> 82

Gln Gln Tyr Asn Asn Tyr Pro Leu Thr

1 5

<210> 83

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3323, TPP-3187X1-hIgG2, VH

<400> 83

caagtgaccc tgaaagagtc cggccctggc atcctgcagc cttcccagac cctgtccctg 60

acctgctcct tctccggctt ctccctgacc acctacggca tcggcgtggg ctggatcaga 120

cagccttctg gcaagggcct ggaatggctg gcccacatct ggtggaacga caacaagtac 180

tacaacaccg ccctgaagtc ccggctgacc atctccaagg acacctccaa caaccaggtg 240

ttcctgaaga tcgcctccgt ggacaccgcc gataccgcca cctactactg cgcccggatc 300

tccctgccct acttcgacta ttggggccag ggcaccaccc tgaccgtcag ctca 354

<210> 84

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3323, TPP-3187X1-hIgG2, VL

<400> 84

gacatcgtga tgacccagtc ccagaaattc atgtccacct ccgtgggcga ccgggtgtcc 60

atcacatgca aggcctctca gaacgtgggc accgccgtgg cctggtatca gcagaagcct 120

ggccagtccc ccaagctgct gatctactcc gcctccaacc ggtacaccgg cgtgcccgat 180

agattcaccg gctctggctc tggcaccgac ttcaccctga ccatctccaa catgcagtcc 240

gaggacctgg ccgactactt ctgccagcag tacaacaact accccctgac cttcggcgct 300

ggcaccaagc tggaactgaa g 321

<210> 85

<211> 443

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3323, TPP-3187X1-hIgG2, heavy chain

<400> 85

Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Thr Thr Tyr

20 25 30

Gly Ile Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Asn Thr Ala

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Asn Asn Gln Val

65 70 75 80

Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Ser Leu Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys

210 215 220

Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr

290 295 300

Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440

<210> 86

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3323, TPP-3187X1-hIgG2, light chain

<400> 86

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Met Gln Ser

65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Asn Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 87

<211> 1329

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3323, TPP-3187X1-hIgG2, heavy chain

<400> 87

caagtgaccc tgaaagagtc cggccctggc atcctgcagc cttcccagac cctgtccctg 60

acctgctcct tctccggctt ctccctgacc acctacggca tcggcgtggg ctggatcaga 120

cagccttctg gcaagggcct ggaatggctg gcccacatct ggtggaacga caacaagtac 180

tacaacaccg ccctgaagtc ccggctgacc atctccaagg acacctccaa caaccaggtg 240

ttcctgaaga tcgcctccgt ggacaccgcc gataccgcca cctactactg cgcccggatc 300

tccctgccct acttcgacta ttggggccag ggcaccaccc tgaccgtcag ctcagcttcc 360

accaagggcc cctccgtgtt ccctctggcc ccttgctccc ggtccacctc tgagtctacc 420

gccgctctgg gctgcctggt gaaagactac ttccccgagc ccgtgaccgt gtcctggaac 480

tctggcgccc tgacctccgg cgtgcacacc tttccagccg tgctgcagtc ctccggcctg 540

tactccctgt cctccgtggt gacagtgccc tcctccaact tcggcaccca gacctacacc 600

tgtaacgtgg accacaagcc ctccaacacc aaggtggaca agaccgtgga acggaagtgc 660

tgcgtggaat gcccaccctg tcctgctcca cctgtggctg gccccagcgt gttcctgttc 720

cccccaaagc ccaaggacac cctgatgatc tcccggaccc ccgaagtgac ctgcgtggtg 780

gtggacgtgt cccacgagga ccccgaggtg cagttcaatt ggtacgtgga cggcgtggaa 840

gtgcacaacg ccaagaccaa gcccagagag gaacagttca actccacctt ccgggtggtg 900

tccgtgctga ccgtggtgca tcaggactgg ctgaacggca aagagtacaa gtgcaaggtc 960

tccaacaagg gcctgcctgc ccccatcgaa aagaccatca gcaagaccaa gggccagccc 1020

cgcgagcccc aggtgtacac actgcccccc agccgggaag agatgaccaa gaaccaggtg 1080

tccctgacct gtctggtgaa aggcttctac ccctccgaca ttgccgtgga atgggagtcc 1140

aacggacagc ctgagaacaa ctacaagacc accccccccca tgctggactc cgacggctca 1200

ttcttcctgt actccaagct gacagtggac aagtcccggt ggcagcaggg caacgtgttc 1260

tcctgctccg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgtccctg 1320

agccccggc 1329

<210> 88

<211> 642

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3323, TPP-3187X1-hIgG2, light chain

<400> 88

gacatcgtga tgacccagtc ccagaaattc atgtccacct ccgtgggcga ccgggtgtcc 60

atcacatgca aggcctctca gaacgtgggc accgccgtgg cctggtatca gcagaagcct 120

ggccagtccc ccaagctgct gatctactcc gcctccaacc ggtacaccgg cgtgcccgat 180

agattcaccg gctctggctc tggcaccgac ttcaccctga ccatctccaa catgcagtcc 240

gaggacctgg ccgactactt ctgccagcag tacaacaact accccctgac cttcggcgct 300

ggcaccaagc tggaactgaa gagaaccgtg gccgctccct ccgtgtttat cttcccaccc 360

tccgacgagc agctgaagtc cggcacagct tccgtcgtgt gcctgctgaa caacttctac 420

ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caactcccag 480

gaatccgtga ccgagcagga ctccaaggac agcacctact ccctgtcctc taccctgacc 540

ctgtccaagg ccgattacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600

ctgtctagcc ccgtgaccaa gtctttcaac cggggcgagt gt 642

<210> 89

<211> 446

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3688, h16C3-hIgG2Kappa, heavy chain

<400> 89

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Leu Ile Ser Thr Tyr Ser Gly Asp Thr Lys Tyr Asn Gln Asn Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Asp Tyr Ser Gly Ser Arg Tyr Trp Phe Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys

210 215 220

Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser

290 295 300

Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 90

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3688, h16C3-hIgG2Kappa, light chain

<400> 90

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Asn Ile Tyr Gly Ala

20 25 30

Leu Asn Trp Tyr Gln Arg Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Asn Leu Ala Thr Gly Met Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Val Leu Ser Ser Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 91

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3705, 090E-M007-A09-Mat1-hIgG2-hIgG2Kappa, VH

<400> 91

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Leu Tyr

20 25 30

Gln Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Trp Ile Ser Phe Ser Gly Gly Asn Thr Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Thr Gly Tyr Ser Ser Pro Trp Tyr Leu Asp Pro Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 92

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3705, 090E-M007-A09-Mat1-hIgG2-hIgG2Kappa, HCDR1

<400> 92

Leu Tyr Gln Met His

1 5

<210> 93

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3705, 090E-M007-A09-Mat1-hIgG2-hIgG2Kappa, HCDR2

<400> 93

Trp Ile Ser Phe Ser Gly Gly Asn Thr Gly Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 94

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3705, 090E-M007-A09-Mat1-hIgG2-hIgG2Kappa, HCDR3

<400> 94

Ala Thr Gly Tyr Ser Ser Pro Trp Tyr Leu Asp Pro

1 5 10

<210> 95

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3705, 090E-M007-A09-Mat1-hIgG2-hIgG2Kappa, VL

<400> 95

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser His Glu Ile Asp Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Tyr Trp Leu Lys Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gly Tyr Asp Asp Leu Ser Val

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys

100 105

<210> 96

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3705, 090E-M007-A09-Mat1-hIgG2-hIgG2Kappa, LCDR1

<400> 96

Gln Ala Ser His Glu Ile Asp Asn Tyr Leu Asn

1 5 10

<210> 97

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3705, 090E-M007-A09-Mat1-hIgG2-hIgG2Kappa, LCDR2

<400> 97

Asp Ala Tyr Trp Leu Lys Thr

1 5

<210> 98

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3705, 090E-M007-A09-Mat1-hIgG2-hIgG2Kappa, LCDR3

<400> 98

Gln Gly Tyr Asp Asp Leu Ser Val Thr

1 5

<210> 99

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3705, 090E-M007-A09-Mat1-hIgG2-hIgG2Kappa, VH

<400> 99

gaagtgcagc tggtggaatc cggcggaggc ctggtgcagc ctggcggatc tctgagactg 60

tcttgtgccg cctccggctt caccttcagc ctgtaccaga tgcactgggt gcgacaggcc 120

cctggcaagg gactggaatg ggtgtcctgg atctccttct ccggcggcaa taccggctac 180

gccgactccg tgaagggccg gttcaccatc tcccgggaca actccaagaa caccctgtac 240

ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgtgc tagagccacc 300

ggctactcct ccccctggta tctggatcct tggggccagg gcacactcgt gaccgtcagc 360

tca 363

<210> 100

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3705, 090E-M007-A09-Mat1-hIgG2-hIgG2Kappa, VL

<400> 100

gacatccaga tgacccagag cccttccagc ctgtccgcct ctgtgggcga cagagtgacc 60

atcacctgtc aggcctccca cgagatcgac aactacctga actggtatca gcagaagccc 120

ggcaaggccc ccaagctgct gatctacgat gcctactggc tgaaaaccgg cgtgccctcc 180

agattctccg gctctggctc tggcaccgac tttaccctga ccatctccag cctgcagccc 240

gaggatatcg ccacctacta ttgtcagggc tacgacgacc tgtccgtgac ctttggcgga 300

ggcaccaagg tggacatcaa g 321

<210> 101

<211> 446

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3705, 090E-M007-A09-Mat1-hIgG2-hIgG2Kappa, heavy chain

<400> 101

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Leu Tyr

20 25 30

Gln Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Trp Ile Ser Phe Ser Gly Gly Asn Thr Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Thr Gly Tyr Ser Ser Pro Trp Tyr Leu Asp Pro Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys

210 215 220

Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser

290 295 300

Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 102

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3705, 090E-M007-A09-Mat1-hIgG2-hIgG2Kappa, light chain

<400> 102

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser His Glu Ile Asp Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Tyr Trp Leu Lys Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gly Tyr Asp Asp Leu Ser Val

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 103

<211> 1338

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3705, 090E-M007-A09-Mat1-hIgG2-hIgG2Kappa, heavy chain

<400> 103

gaagtgcagc tggtggaatc cggcggaggc ctggtgcagc ctggcggatc tctgagactg 60

tcttgtgccg cctccggctt caccttcagc ctgtaccaga tgcactgggt gcgacaggcc 120

cctggcaagg gactggaatg ggtgtcctgg atctccttct ccggcggcaa taccggctac 180

gccgactccg tgaagggccg gttcaccatc tcccgggaca actccaagaa caccctgtac 240

ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgtgc tagagccacc 300

ggctactcct ccccctggta tctggatcct tggggccagg gcacactcgt gaccgtcagc 360

tcagcttcca ccaagggccc ctccgtgttc cctctggccc cttgctcccg gtccacctct 420

gagtctaccg ccgctctggg ctgcctggtg aaagactact tccccgagcc cgtgaccgtg 480

tcctggaact ctggcgccct gacctccggc gtgcacacct ttccagccgt gctgcagtcc 540

tccggcctgt actccctgtc ctccgtggtg acagtgccct cctccaactt cggcacccag 600

acctacacct gtaacgtgga ccacaagccc tccaacacca aggtggacaa gaccgtggaa 660

cggaagtgct gcgtggaatg cccaccctgt cctgctccac ctgtggctgg ccccagcgtg 720

ttcctgttcc ccccaaagcc caaggacacc ctgatgatct cccggacccc cgaagtgacc 780

tgcgtggtgg tggacgtgtc ccacgaggac cccgaggtgc agttcaattg gtacgtggac 840

ggcgtggaag tgcacaacgc caagaccaag cccagagagg aacagttcaa ctccaccttc 900

cgggtggtgt ccgtgctgac cgtggtgcat caggactggc tgaacggcaa agagtacaag 960

tgcaaggtct ccaacaaggg cctgcctgcc cccatcgaaa agaccatcag caagaccaag 1020

ggccagcccc gcgagcccca ggtgtacaca ctgcccccca gccgggaaga gatgaccaag 1080

aaccaggtgt ccctgacctg tctggtgaaa ggcttctacc cctccgacat tgccgtggaa 1140

tgggagtcca acggacagcc tgagaacaac tacaagacca ccccccccat gctggactcc 1200

gacggctcat tcttcctgta ctccaagctg acagtggaca agtcccggtg gcagcagggc 1260

aacgtgttct cctgctccgt gatgcacgag gccctgcaca accactacac ccagaagtcc 1320

ctgtccctga gccccggc 1338

<210> 104

<211> 642

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3705, 090E-M007-A09-Mat1-hIgG2-hIgG2Kappa, light chain

<400> 104

gacatccaga tgacccagag cccttccagc ctgtccgcct ctgtgggcga cagagtgacc 60

atcacctgtc aggcctccca cgagatcgac aactacctga actggtatca gcagaagccc 120

ggcaaggccc ccaagctgct gatctacgat gcctactggc tgaaaaccgg cgtgccctcc 180

agattctccg gctctggctc tggcaccgac tttaccctga ccatctccag cctgcagccc 240

gaggatatcg ccacctacta ttgtcagggc tacgacgacc tgtccgtgac ctttggcgga 300

ggcaccaagg tggacatcaa gcggacagtg gccgctccct ccgtgtttat cttcccaccc 360

tccgacgagc agctgaagtc cggcacagct tccgtcgtgt gcctgctgaa caacttctac 420

ccccgcgagg ccaaggtgca gtggaaagtg gataacgccc tgcagtccgg caactcccag 480

gaatccgtga ccgagcagga ctccaaggac agcacctact ccctgtcctc caccctgacc 540

ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600

ctgtctagcc ccgtgaccaa gtctttcaac cggggcgagt gt 642

<210> 105

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3707, 090E-M007-A09-Mat2-hIgG2-hIgG2Kappa, VH

<400> 105

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Leu Tyr

20 25 30

Gln Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Trp Ile Ser Phe Ser Gly Gly Asn Thr Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Thr Gly Tyr Ser Ser Pro Trp Tyr Leu Asp Pro Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 106

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3707, 090E-M007-A09-Mat2-hIgG2-hIgG2Kappa, HCDR1

<400> 106

Leu Tyr Gln Met His

1 5

<210> 107

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3707, 090E-M007-A09-Mat2-hIgG2-hIgG2Kappa, HCDR2

<400> 107

Trp Ile Ser Phe Ser Gly Gly Asn Thr Gly Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 108

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3707, 090E-M007-A09-Mat2-hIgG2-hIgG2Kappa, HCDR3

<400> 108

Ala Thr Gly Tyr Ser Ser Pro Trp Tyr Leu Asp Pro

1 5 10

<210> 109

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3707, 090E-M007-A09-Mat2-hIgG2-hIgG2Kappa, VL

<400> 109

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser His Glu Ile Asp Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Tyr Trp Ser Lys Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gly Tyr Asp Asp Leu Ser Val

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys

100 105

<210> 110

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3707, 090E-M007-A09-Mat2-hIgG2-hIgG2Kappa, LCDR1

<400> 110

Gln Ala Ser His Glu Ile Asp Asn Tyr Leu Asn

1 5 10

<210> 111

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3707, 090E-M007-A09-Mat2-hIgG2-hIgG2Kappa, LCDR2

<400> 111

Asp Ala Tyr Trp Ser Lys Thr

1 5

<210> 112

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3707, 090E-M007-A09-Mat2-hIgG2-hIgG2Kappa, LCDR3

<400> 112

Gln Gly Tyr Asp Asp Leu Ser Val Thr

1 5

<210> 113

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3707, 090E-M007-A09-Mat2-hIgG2-hIgG2Kappa, VH

<400> 113

gaagtgcagc tggtggaatc cggcggaggc ctggtgcagc ctggcggatc tctgagactg 60

tcttgtgccg cctccggctt caccttcagc ctgtaccaga tgcactgggt gcgacaggcc 120

cctggcaagg gactggaatg ggtgtcctgg atctccttct ccggcggcaa taccggctac 180

gccgactccg tgaagggccg gttcaccatc tcccgggaca actccaagaa caccctgtac 240

ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgtgc tagagccacc 300

ggctactcct ccccctggta tctggatcct tggggccagg gcacactcgt gaccgtcagc 360

tca 363

<210> 114

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3707, 090E-M007-A09-Mat2-hIgG2-hIgG2Kappa, VL

<400> 114

gacatccaga tgacccagag cccttccagc ctgtccgcct ctgtgggcga cagagtgacc 60

atcacctgtc aggcctccca cgagatcgac aactacctga actggtatca gcagaagccc 120

ggcaaggccc ccaagctgct gatctacgac gcctactggt ccaagaccgg cgtgccctcc 180

agattctccg gctctggctc tggcaccgac tttaccctga ccatctccag cctgcagccc 240

gaggatatcg ccacctacta ttgtcagggc tacgacgacc tgtccgtgac ctttggcgga 300

ggcaccaagg tggacatcaa g 321

<210> 115

<211> 446

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3707, 090E-M007-A09-Mat2-hIgG2-hIgG2Kappa, heavy chain

<400> 115

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Leu Tyr

20 25 30

Gln Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Trp Ile Ser Phe Ser Gly Gly Asn Thr Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Thr Gly Tyr Ser Ser Pro Trp Tyr Leu Asp Pro Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys

210 215 220

Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser

290 295 300

Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 116

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3707, 090E-M007-A09-Mat2-hIgG2-hIgG2Kappa, light chain

<400> 116

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser His Glu Ile Asp Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Tyr Trp Ser Lys Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gly Tyr Asp Asp Leu Ser Val

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 117

<211> 1338

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3707, 090E-M007-A09-Mat2-hIgG2-hIgG2Kappa, heavy chain

<400> 117

gaagtgcagc tggtggaatc cggcggaggc ctggtgcagc ctggcggatc tctgagactg 60

tcttgtgccg cctccggctt caccttcagc ctgtaccaga tgcactgggt gcgacaggcc 120

cctggcaagg gactggaatg ggtgtcctgg atctccttct ccggcggcaa taccggctac 180

gccgactccg tgaagggccg gttcaccatc tcccgggaca actccaagaa caccctgtac 240

ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgtgc tagagccacc 300

ggctactcct ccccctggta tctggatcct tggggccagg gcacactcgt gaccgtcagc 360

tcagcttcca ccaagggccc ctccgtgttc cctctggccc cttgctcccg gtccacctct 420

gagtctaccg ccgctctggg ctgcctggtg aaagactact tccccgagcc cgtgaccgtg 480

tcctggaact ctggcgccct gacctccggc gtgcacacct ttccagccgt gctgcagtcc 540

tccggcctgt actccctgtc ctccgtggtg acagtgccct cctccaactt cggcacccag 600

acctacacct gtaacgtgga ccacaagccc tccaacacca aggtggacaa gaccgtggaa 660

cggaagtgct gcgtggaatg cccaccctgt cctgctccac ctgtggctgg ccccagcgtg 720

ttcctgttcc ccccaaagcc caaggacacc ctgatgatct cccggacccc cgaagtgacc 780

tgcgtggtgg tggacgtgtc ccacgaggac cccgaggtgc agttcaattg gtacgtggac 840

ggcgtggaag tgcacaacgc caagaccaag cccagagagg aacagttcaa ctccaccttc 900

cgggtggtgt ccgtgctgac cgtggtgcat caggactggc tgaacggcaa agagtacaag 960

tgcaaggtct ccaacaaggg cctgcctgcc cccatcgaaa agaccatcag caagaccaag 1020

ggccagcccc gcgagcccca ggtgtacaca ctgcccccca gccgggaaga gatgaccaag 1080

aaccaggtgt ccctgacctg tctggtgaaa ggcttctacc cctccgacat tgccgtggaa 1140

tgggagtcca acggacagcc tgagaacaac tacaagacca ccccccccat gctggactcc 1200

gacggctcat tcttcctgta ctccaagctg acagtggaca agtcccggtg gcagcagggc 1260

aacgtgttct cctgctccgt gatgcacgag gccctgcaca accactacac ccagaagtcc 1320

ctgtccctga gccccggc 1338

<210> 118

<211> 642

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3707, 090E-M007-A09-Mat2-hIgG2-hIgG2Kappa, light chain

<400> 118

gacatccaga tgacccagag cccttccagc ctgtccgcct ctgtgggcga cagagtgacc 60

atcacctgtc aggcctccca cgagatcgac aactacctga actggtatca gcagaagccc 120

ggcaaggccc ccaagctgct gatctacgac gcctactggt ccaagaccgg cgtgccctcc 180

agattctccg gctctggctc tggcaccgac tttaccctga ccatctccag cctgcagccc 240

gaggatatcg ccacctacta ttgtcagggc tacgacgacc tgtccgtgac ctttggcgga 300

ggcaccaagg tggacatcaa gcggacagtg gccgctccct ccgtgtttat cttcccaccc 360

tccgacgagc agctgaagtc cggcacagct tccgtcgtgt gcctgctgaa caacttctac 420

ccccgcgagg ccaaggtgca gtggaaagtg gataacgccc tgcagtccgg caactcccag 480

gaatccgtga ccgagcagga ctccaaggac agcacctact ccctgtcctc caccctgacc 540

ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600

ctgtctagcc ccgtgaccaa gtctttcaac cggggcgagt gt 642

<210> 119

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3714, TPP-2971HU2-hIgG2Kappa, VH

<400> 119

Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Tyr

20 25 30

Gly Ile Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Ser Thr Ser

50 55 60

Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Ser Leu Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 120

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3714, TPP-2971HU2-hIgG2Kappa, HCDR1

<400> 120

Thr Tyr Gly Ile Gly Val Gly

1 5

<210> 121

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3714, TPP-2971HU2-hIgG2Kappa, HCDR2

<400> 121

His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Ser Thr Ser Leu Lys Thr

1 5 10 15

<210> 122

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3714, TPP-2971HU2-hIgG2Kappa, HCDR3

<400> 122

Ile Ser Leu Pro Tyr Phe Asp Tyr

1 5

<210> 123

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3714, TPP-2971HU2-hIgG2Kappa, VL

<400> 123

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 124

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3714, TPP-2971HU2-hIgG2Kappa, LCDR1

<400> 124

Lys Ala Ser Gln Asn Val Gly Thr Ala Val Ala

1 5 10

<210> 125

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3714, TPP-2971HU2-hIgG2Kappa, LCDR2

<400> 125

Ser Ala Ser Asn Arg Tyr Thr

1 5

<210> 126

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3714, TPP-2971HU2-hIgG2Kappa, LCDR3

<400> 126

Gln Gln Tyr Ser Ser Tyr Pro Leu Thr

1 5

<210> 127

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3714, TPP-2971HU2-hIgG2Kappa, VH

<400> 127

caagtgaccc tgagagagtc cggccctgcc ctcgtgaagc ctacccagac cctgacactg 60

acctgcacct tctccggctt ctccctgtcc acctacggca tcggcgtggg ctggatcaga 120

cagcctcctg gcaaggccct ggaatggctg gctcacatct ggtggaacga caacaagtac 180

tactccacct ccctgaaaac ccggctgacc atctccaagg acacctccaa gaaccaggtg 240

gtgctgacca tgaccaacat ggaccccgtg gacaccgcca cctactactg cgccagaatc 300

tccctgccct acttcgacta ctggggccag ggcacactcg tgaccgtcag ctca 354

<210> 128

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3714, TPP-2971HU2-hIgG2Kappa, VL

<400> 128

gatatccagc tgacccagtc ccccagcttc ctgtctgcct ctgtgggcga cagagtgacc 60

atcacatgca aggcctccca gaacgtgggc accgccgtgg cttggtatca gcagaagcct 120

ggcaaggccc ccaagctgct gatctactcc gcctccaacc ggtacaccgg cgtgccctct 180

agattctccg gctctggctc tggcaccgag tttaccctga ccatctccag cctgcagccc 240

gaggacttcg ccacctacta ctgccagcag tactcctcct accccctgac ctttggcgga 300

ggcaccaagg tggaaatcaa g 321

<210> 129

<211> 443

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3714, TPP-2971HU2-hIgG2Kappa, heavy chain

<400> 129

Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Tyr

20 25 30

Gly Ile Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Ser Thr Ser

50 55 60

Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Ser Leu Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys

210 215 220

Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr

290 295 300

Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440

<210> 130

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3714, TPP-2971HU2-hIgG2Kappa, light chain

<400> 130

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 131

<211> 1329

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3714, TPP-2971HU2-hIgG2Kappa, heavy chain

<400> 131

caagtgaccc tgagagagtc cggccctgcc ctcgtgaagc ctacccagac cctgacactg 60

acctgcacct tctccggctt ctccctgtcc acctacggca tcggcgtggg ctggatcaga 120

cagcctcctg gcaaggccct ggaatggctg gctcacatct ggtggaacga caacaagtac 180

tactccacct ccctgaaaac ccggctgacc atctccaagg acacctccaa gaaccaggtg 240

gtgctgacca tgaccaacat ggaccccgtg gacaccgcca cctactactg cgccagaatc 300

tccctgccct acttcgacta ctggggccag ggcacactcg tgaccgtcag ctcagcttcc 360

accaagggcc cctccgtgtt ccctctggcc ccttgctccc ggtccacctc tgagtctacc 420

gccgctctgg gctgcctggt gaaagactac ttccccgagc ccgtgaccgt gtcctggaac 480

tctggcgccc tgacctccgg cgtgcacacc tttccagccg tgctgcagtc ctccggcctg 540

tactccctgt cctccgtggt gacagtgccc tcctccaact tcggcaccca gacctacacc 600

tgtaacgtgg accacaagcc ctccaacacc aaggtggaca agaccgtgga acggaagtgc 660

tgcgtggaat gcccaccctg tcctgctcca cctgtggctg gccccagcgt gttcctgttc 720

cccccaaagc ccaaggacac cctgatgatc tcccggaccc ccgaagtgac ctgcgtggtg 780

gtggacgtgt cccacgagga ccccgaggtg cagttcaatt ggtacgtgga cggcgtggaa 840

gtgcacaacg ccaagaccaa gcccagagag gaacagttca actccacctt ccgggtggtg 900

tccgtgctga ccgtggtgca tcaggactgg ctgaacggca aagagtacaa gtgcaaggtc 960

tccaacaagg gcctgcctgc ccccatcgaa aagaccatca gcaagaccaa gggccagccc 1020

cgcgagcccc aggtgtacac actgcccccc agccgggaag agatgaccaa gaaccaggtg 1080

tccctgacct gtctggtgaa aggcttctac ccctccgaca ttgccgtgga atgggagtcc 1140

aacggacagc ctgagaacaa ctacaagacc accccccccca tgctggactc cgacggctca 1200

ttcttcctgt actccaagct gacagtggac aagtcccggt ggcagcaggg caacgtgttc 1260

tcctgctccg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgtccctg 1320

agccccggc 1329

<210> 132

<211> 642

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3714, TPP-2971HU2-hIgG2Kappa, light chain

<400> 132

gatatccagc tgacccagtc ccccagcttc ctgtctgcct ctgtgggcga cagagtgacc 60

atcacatgca aggcctccca gaacgtgggc accgccgtgg cttggtatca gcagaagcct 120

ggcaaggccc ccaagctgct gatctactcc gcctccaacc ggtacaccgg cgtgccctct 180

agattctccg gctctggctc tggcaccgag tttaccctga ccatctccag cctgcagccc 240

gaggacttcg ccacctacta ctgccagcag tactcctcct accccctgac ctttggcgga 300

ggcaccaagg tggaaatcaa gcggaccgtg gccgctccct ccgtgtttat cttcccaccc 360

tccgacgagc agctgaagtc cggcacagct tccgtcgtgt gcctgctgaa caacttctac 420

ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg caactcccag 480

gaatccgtga ccgagcagga ctccaaggac agcacctact ccctgtcctc caccctgacc 540

ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600

ctgtctagcc ccgtgaccaa gtctttcaac cggggcgagt gt 642

<210> 133

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3820, 3187HU1-hIgG2Kappa, VH

<400> 133

Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Thr Thr Tyr

20 25 30

Gly Ile Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Ser Thr Ser

50 55 60

Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Ser Leu Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 134

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3820, 3187HU1-hIgG2Kappa, HCDR1

<400> 134

Thr Tyr Gly Ile Gly Val Gly

1 5

<210> 135

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3820, 3187HU1-hIgG2Kappa, HCDR2

<400> 135

His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Ser Thr Ser Leu Lys Thr

1 5 10 15

<210> 136

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3820, 3187HU1-hIgG2Kappa, HCDR3

<400> 136

Ile Ser Leu Pro Tyr Phe Asp Tyr

1 5

<210> 137

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3820, 3187HU1-hIgG2Kappa, VL

<400> 137

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 138

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3820, 3187HU1-hIgG2Kappa, LCDR1

<400> 138

Lys Ala Ser Gln Asn Val Gly Thr Ala Val Ala

1 5 10

<210> 139

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3820, 3187HU1-hIgG2Kappa, LCDR2

<400> 139

Ser Ala Ser Asn Arg Tyr Thr

1 5

<210> 140

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3820, 3187HU1-hIgG2Kappa, LCDR3

<400> 140

Gln Gln Tyr Asn Asn Tyr Pro Leu Thr

1 5

<210> 141

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3820, 3187HU1-hIgG2Kappa, VH

<400> 141

caagtgaccc tgagagagtc cggccctgcc ctcgtgaagc ctacccagac cctgacactg 60

acctgcacct tcagcggctt cagcctgacc acctacggca tcggcgtggg ctggatcaga 120

cagcctcctg gcaagggcct ggaatggctg gcccacatct ggtggaacga caacaagtac 180

tacagcacca gcctgaaaac ccggctgacc atcagcaagg acaccagcaa gaaccaggtg 240

gtgctgacca tgaccaacat ggaccccgtg gacaccgcca cctactactg cgccagaatc 300

agcctgccct acttcgacta ctggggccag ggcaccctcg tgacagtgtc atca 354

<210> 142

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3820, 3187HU1-hIgG2Kappa, VL

<400> 142

gatatccagc tgacccagag ccccagcttt ctgagcgcca gcgtgggcga cagagtgacc 60

atcacatgca aggccagcca gaacgtgggc acagccgtgg cctggtatca gcagaagcct 120

ggcaaggccc ccaagctgct gatctacagc gccagcaacc ggtacaccgg cgtgcccagc 180

agattttctg gcagcggctc cggcaccgag ttcaccctga caatcagcag cctgcagccc 240

gaggacttcg ccacctacta ctgccagcag tacaacaact accccctgac cttcggcgga 300

ggcaccaagg tggaaattaa a 321

<210> 143

<211> 443

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3820, 3187HU1-hIgG2Kappa, heavy chain

<400> 143

Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Thr Thr Tyr

20 25 30

Gly Ile Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Ser Thr Ser

50 55 60

Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Ser Leu Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys

210 215 220

Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr

290 295 300

Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440

<210> 144

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3820, 3187HU1-hIgG2Kappa, light chain

<400> 144

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 145

<211> 1329

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3820, 3187HU1-hIgG2Kappa, heavy chain

<400> 145

caagtgaccc tgagagagtc cggccctgcc ctcgtgaagc ctacccagac cctgacactg 60

acctgcacct tcagcggctt cagcctgacc acctacggca tcggcgtggg ctggatcaga 120

cagcctcctg gcaagggcct ggaatggctg gcccacatct ggtggaacga caacaagtac 180

tacagcacca gcctgaaaac ccggctgacc atcagcaagg acaccagcaa gaaccaggtg 240

gtgctgacca tgaccaacat ggaccccgtg gacaccgcca cctactactg cgccagaatc 300

agcctgccct acttcgacta ctggggccag ggcaccctcg tgacagtgtc atcagccagc 360

accaagggcc ccagcgtgtt ccctctggcc ccttgtagca gaagcaccag cgagtctaca 420

gccgccctgg gctgcctcgt gaaggactac tttcccgagc ccgtgaccgt gtcctggaac 480

tctggcgctc tgacaagcgg cgtgcacacc tttccagccg tgctgcagag cagcggcctg 540

tactctctga gcagcgtcgt gacagtgccc agcagcaact tcggcaccca gacctacacc 600

tgtaacgtgg accacaagcc cagcaacacc aaggtggaca agaccgtgga acggaagtgc 660

tgcgtggaat gccccccttg tcctgcccct ccagtggctg gcccttccgt gttcctgttc 720

cccccaaagc ccaaggacac cctgatgatc agccggaccc ccgaagtgac ctgcgtggtg 780

gtggatgtgt cccacgagga ccccgaggtg cagttcaatt ggtacgtgga cggcgtggaa 840

gtgcacaacg ccaagaccaa gcccagagag gaacagttca acagcacctt ccgggtggtg 900

tccgtgctga ccgtggtgca tcaggactgg ctgaacggca aagagtacaa gtgcaaggtg 960

tccaacaagg gcctgcctgc ccccatcgag aaaaccatca gcaagaccaa aggccagccc 1020

cgcgagcccc aggtgtacac actgcctcca agccgggaag agatgaccaa gaaccaggtg 1080

tccctgacct gtctcgtgaa aggcttctac ccctccgata tcgccgtgga atgggagagc 1140

aacggccagc ccgagaacaa ctacaagacc accccccccca tgctggacag cgacggctca 1200

ttcttcctgt acagcaagct gacagtggac aagtcccggt ggcagcaggg caacgtgttc 1260

agctgcagcg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgagcctg 1320

agccctggc 1329

<210> 146

<211> 642

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3820, 3187HU1-hIgG2Kappa, light chain

<400> 146

gatatccagc tgacccagag ccccagcttt ctgagcgcca gcgtgggcga cagagtgacc 60

atcacatgca aggccagcca gaacgtgggc acagccgtgg cctggtatca gcagaagcct 120

ggcaaggccc ccaagctgct gatctacagc gccagcaacc ggtacaccgg cgtgcccagc 180

agattttctg gcagcggctc cggcaccgag ttcaccctga caatcagcag cctgcagccc 240

gaggacttcg ccacctacta ctgccagcag tacaacaact accccctgac cttcggcgga 300

ggcaccaagg tggaaattaa acgaaccgtg gccgctccca gcgtgttcat cttcccacct 360

agcgacgagc agctgaagtc cggcacagcc tctgtcgtgt gcctgctgaa caacttctac 420

ccccgcgagg ccaaggtgca gtggaaggtg gacaatgccc tgcagagcgg caacagccag 480

gaaagcgtga ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540

ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600

ctgtctagcc ccgtgaccaa gagcttcaac cggggcgagt gt 642

<210> 147

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3821, 3187HU2-hIgG2Kappa, VH

<400> 147

Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Tyr

20 25 30

Gly Ile Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Ser Thr Ser

50 55 60

Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Ser Leu Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 148

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3821, 3187HU2-hIgG2Kappa, HCDR1

<400> 148

Thr Tyr Gly Ile Gly Val Gly

1 5

<210> 149

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3821, 3187HU2-hIgG2Kappa, HCDR2

<400> 149

His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Ser Thr Ser Leu Lys Thr

1 5 10 15

<210> 150

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3821, 3187HU2-hIgG2Kappa, HCDR3

<400> 150

Ile Ser Leu Pro Tyr Phe Asp Tyr

1 5

<210> 151

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3821, 3187HU2-hIgG2Kappa, VL

<400> 151

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 152

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3821, 3187HU2-hIgG2Kappa, LCDR1

<400> 152

Lys Ala Ser Gln Asn Val Gly Thr Ala Val Ala

1 5 10

<210> 153

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3821, 3187HU2-hIgG2Kappa, LCDR2

<400> 153

Ser Ala Ser Asn Arg Tyr Thr

1 5

<210> 154

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3821, 3187HU2-hIgG2Kappa, LCDR3

<400> 154

Gln Gln Tyr Asn Asn Tyr Pro Leu Thr

1 5

<210> 155

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3821, 3187HU2-hIgG2Kappa, VH

<400> 155

caggtcacac tgagagagtc cggccctgcc ctggtgaaac ccacccagac cctgaccctg 60

acatgcacct tcagcggctt cagcctgagc acctacggca tcggcgtggg ctggatcaga 120

cagccccctg gcaaggccct ggaatggctg gcccacatct ggtggaacga caacaagtac 180

tacagcacca gcctgaaaac ccggctgacc atcagcaagg acaccagcaa gaaccaggtg 240

gtgctgacca tgaccaacat ggaccccgtg gacaccgcca cctactactg cgcccggatc 300

agcctgccct acttcgacta ctggggccag ggcaccctgg tgaccgtgtc ctca 354

<210> 156

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3821, 3187HU2-hIgG2Kappa, VL

<400> 156

gatatccagc tgacccagag ccccagcttt ctgagcgcca gcgtgggcga cagagtgacc 60

atcacatgca aggccagcca gaacgtgggc acagccgtgg cctggtatca gcagaagcct 120

ggcaaggccc ccaagctgct gatctacagc gccagcaacc ggtacaccgg cgtgcccagc 180

agattttctg gcagcggctc cggcaccgag ttcaccctga caatcagcag cctgcagccc 240

gaggacttcg ccacctacta ctgccagcag tacaacaact accccctgac cttcggcgga 300

ggcaccaagg tggaaattaa a 321

<210> 157

<211> 443

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3821, 3187HU2-hIgG2Kappa, heavy chain

<400> 157

Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Tyr

20 25 30

Gly Ile Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Ser Thr Ser

50 55 60

Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Ser Leu Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys

210 215 220

Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr

290 295 300

Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440

<210> 158

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-3821, 3187HU2-hIgG2Kappa, light chain

<400> 158

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 159

<211> 1329

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3821, 3187HU2-hIgG2Kappa, heavy chain

<400> 159

caggtcacac tgagagagtc cggccctgcc ctggtgaaac ccacccagac cctgaccctg 60

acatgcacct tcagcggctt cagcctgagc acctacggca tcggcgtggg ctggatcaga 120

cagccccctg gcaaggccct ggaatggctg gcccacatct ggtggaacga caacaagtac 180

tacagcacca gcctgaaaac ccggctgacc atcagcaagg acaccagcaa gaaccaggtg 240

gtgctgacca tgaccaacat ggaccccgtg gacaccgcca cctactactg cgcccggatc 300

agcctgccct acttcgacta ctggggccag ggcaccctgg tgaccgtgtc ctcagccagc 360

accaagggcc ccagcgtgtt ccctctggcc ccttgtagca gaagcaccag cgagtctaca 420

gccgccctgg gctgcctcgt gaaggactac tttcccgagc ccgtgaccgt gtcctggaac 480

tctggcgctc tgacaagcgg cgtgcacacc tttccagccg tgctgcagag cagcggcctg 540

tactctctga gcagcgtcgt gacagtgccc agcagcaact tcggcaccca gacctacacc 600

tgtaacgtgg accacaagcc cagcaacacc aaggtggaca agaccgtgga acggaagtgc 660

tgcgtggaat gccccccttg tcctgcccct ccagtggctg gcccttccgt gttcctgttc 720

cccccaaagc ccaaggacac cctgatgatc agccggaccc ccgaagtgac ctgcgtggtg 780

gtggatgtgt cccacgagga ccccgaggtg cagttcaatt ggtacgtgga cggcgtggaa 840

gtgcacaacg ccaagaccaa gcccagagag gaacagttca acagcacctt ccgggtggtg 900

tccgtgctga ccgtggtgca tcaggactgg ctgaacggca aagagtacaa gtgcaaggtg 960

tccaacaagg gcctgcctgc ccccatcgag aaaaccatca gcaagaccaa aggccagccc 1020

cgcgagcccc aggtgtacac actgcctcca agccgggaag agatgaccaa gaaccaggtg 1080

tccctgacct gtctcgtgaa aggcttctac ccctccgata tcgccgtgga atgggagagc 1140

aacggccagc ccgagaacaa ctacaagacc accccccccca tgctggacag cgacggctca 1200

ttcttcctgt acagcaagct gacagtggac aagtcccggt ggcagcaggg caacgtgttc 1260

agctgcagcg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgagcctg 1320

agccctggc 1329

<210> 160

<211> 642

<212> DNA

<213> Artificial sequence

<220>

<223> TPP-3821, 3187HU2-hIgG2Kappa, light chain

<400> 160

gatatccagc tgacccagag ccccagcttt ctgagcgcca gcgtgggcga cagagtgacc 60

atcacatgca aggccagcca gaacgtgggc acagccgtgg cctggtatca gcagaagcct 120

ggcaaggccc ccaagctgct gatctacagc gccagcaacc ggtacaccgg cgtgcccagc 180

agattttctg gcagcggctc cggcaccgag ttcaccctga caatcagcag cctgcagccc 240

gaggacttcg ccacctacta ctgccagcag tacaacaact accccctgac cttcggcgga 300

ggcaccaagg tggaaattaa acgaaccgtg gccgctccca gcgtgttcat cttcccacct 360

agcgacgagc agctgaagtc cggcacagcc tctgtcgtgt gcctgctgaa caacttctac 420

ccccgcgagg ccaaggtgca gtggaaggtg gacaatgccc tgcagagcgg caacagccag 480

gaaagcgtga ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540

ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600

ctgtctagcc ccgtgaccaa gagcttcaac cggggcgagt gt 642

<210> 161

<211> 532

<212> PRT

<213> Macaca mulatta (Rhesus monkey)

<400> 161

Gln Leu Thr Ile Glu Ser Arg Pro Phe Asn Val Ala Glu Gly Lys Glu

1 5 10 15

Val Leu Leu Leu Ala His Asn Leu Pro Gln Asn Leu Ile Gly Phe Asn

20 25 30

Trp Tyr Lys Gly Glu Arg Val Asp Ala Lys Arg Leu Ile Val Ala Tyr

35 40 45

Val Ile Glu Thr Gln Gln Thr Thr Pro Gly Pro Ala His Ser Gly Arg

50 55 60

Glu Thr Val Tyr Ser Asn Ala Ser Leu Leu Ile Gln Asn Val Thr Gln

65 70 75 80

Asn Asp Thr Gly Ser Tyr Thr Leu Gln Val Ile Lys Gly Asp Leu Val

85 90 95

Asn Glu Glu Ala Thr Gly Arg Phe Trp Val Tyr Pro Glu Leu Pro Lys

100 105 110

Pro Tyr Ile Thr Ser Asn Asn Ser Asn Pro Val Glu Asp Lys Asp Ala

115 120 125

Val Asp Phe Thr Cys Glu Pro Asp Ile His Ser Thr Thr Tyr Leu Trp

130 135 140

Trp Val Asn Asp Gln Ser Leu Pro Val Ser Pro Arg Leu Gln Leu Ser

145 150 155 160

Asn Gly Asn Arg Thr Leu Thr Leu Leu Ser Val Lys Arg Asn Asp Ala

165 170 175

Gly Ala Tyr Glu Cys Glu Ile Gln Asn Pro Val Ser Ala Asn Leu Ser

180 185 190

Asp Pro Val Ile Leu Asn Val Leu Tyr Gly Pro Asp Val Pro Thr Ile

195 200 205

Ser Pro Ser Asn Ser Asn Tyr Arg Pro Gly Glu Asn Leu Asn Leu Ser

210 215 220

Cys His Ala Ala Ser Asn Pro Thr Ala Gln Tyr Ser Trp Phe Val Asn

225 230 235 240

Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn Ile Thr

245 250 255

Val Asn Asn Ser Gly Ser Tyr Met Cys Gln Ala Tyr Asn Ser Ala Thr

260 265 270

Gly Leu Asn Arg Thr Thr Val Met Met Ile Thr Val Ser Gly Ile Glu

275 280 285

Gly Arg Asp Met Asp Thr Gly Pro Lys Ser Cys Asp Lys Thr His Thr

290 295 300

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

305 310 315 320

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

325 330 335

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

340 345 350

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

355 360 365

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

370 375 380

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

385 390 395 400

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

405 410 415

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

420 425 430

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

435 440 445

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

450 455 460

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

465 470 475 480

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

485 490 495

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

500 505 510

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys His His

515 520 525

His His His His

530

<210> 162

<211> 292

<212> PRT

<213> Homo sapiens

<400> 162

Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu Gly Lys Glu

1 5 10 15

Val Leu Leu Leu Ala His Asn Leu Pro Gln Asn Arg Ile Gly Tyr Ser

20 25 30

Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Ser Leu Ile Val Gly Tyr

35 40 45

Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser Gly Arg

50 55 60

Glu Thr Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Val Thr Gln

65 70 75 80

Asn Asp Thr Gly Phe Tyr Thr Leu Gln Val Ile Lys Ser Asp Leu Val

85 90 95

Asn Glu Glu Ala Thr Gly Gln Phe His Val Tyr Pro Glu Leu Pro Lys

100 105 110

Pro Ser Ile Ser Ser Asn Asn Ser Asn Pro Val Glu Asp Lys Asp Ala

115 120 125

Val Ala Phe Thr Cys Glu Pro Glu Val Gln Asn Thr Thr Tyr Leu Trp

130 135 140

Trp Val Asn Gly Gln Ser Leu Pro Val Ser Pro Arg Leu Gln Leu Ser

145 150 155 160

Asn Gly Asn Met Thr Leu Thr Leu Leu Ser Val Lys Arg Asn Asp Ala

165 170 175

Gly Ser Tyr Glu Cys Glu Ile Gln Asn Pro Ala Ser Ala Asn Arg Ser

180 185 190

Asp Pro Val Thr Leu Asn Val Leu Tyr Gly Pro Asp Gly Pro Thr Ile

195 200 205

Ser Pro Ser Lys Ala Asn Tyr Arg Pro Gly Glu Asn Leu Asn Leu Ser

210 215 220

Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Phe Ile Asn

225 230 235 240

Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn Ile Thr

245 250 255

Val Asn Asn Ser Gly Ser Tyr Met Cys Gln Ala His Asn Ser Ala Thr

260 265 270

Gly Leu Asn Arg Thr Thr Val Thr Met Ile Thr Val Ser Gly His His

275 280 285

His His His His

290

<210> 163

<211> 404

<212> PRT

<213> Homo sapiens

<400> 163

Gln Leu Thr Thr Glu Ser Met Pro Phe Asn Val Ala Glu Gly Lys Glu

1 5 10 15

Val Leu Leu Leu Val His Asn Leu Pro Gln Gln Leu Phe Gly Tyr Ser

20 25 30

Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gln Ile Val Gly Tyr

35 40 45

Ala Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Asn Ser Gly Arg

50 55 60

Glu Thr Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Val Thr Gln

65 70 75 80

Asn Asp Thr Gly Phe Tyr Thr Leu Gln Val Ile Lys Ser Asp Leu Val

85 90 95

Asn Glu Glu Ala Thr Gly Gln Phe His Val Tyr Pro Glu Leu Pro Lys

100 105 110

Pro Ser Ile Ser Ser Asn Asn Ser Asn Pro Val Glu Asp Lys Asp Ala

115 120 125

Val Ala Phe Thr Cys Glu Pro Glu Thr Gln Asp Thr Thr Tyr Leu Trp

130 135 140

Trp Ile Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln Leu Ser

145 150 155 160

Asn Gly Asn Arg Thr Leu Thr Leu Leu Ser Val Thr Arg Asn Asp Thr

165 170 175

Gly Pro Tyr Glu Cys Glu Ile Gln Asn Pro Val Ser Ala Asn Arg Ser

180 185 190

Asp Pro Val Thr Leu Asn Val Thr Tyr Gly Pro Asp Thr Pro Thr Ile

195 200 205

Ser Pro Ser Asp Thr Tyr Tyr Arg Pro Gly Ala Asn Leu Ser Leu Ser

210 215 220

Cys Tyr Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Leu Ile Asn

225 230 235 240

Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn Ile Thr

245 250 255

Val Asn Asn Ser Gly Ser Tyr Thr Cys His Ala Asn Asn Ser Val Thr

260 265 270

Gly Cys Asn Arg Thr Thr Val Lys Thr Ile Ile Val Thr Glu Leu Ser

275 280 285

Pro Val Val Ala Lys Pro Gln Ile Lys Ala Ser Lys Thr Thr Val Thr

290 295 300

Gly Asp Lys Asp Ser Val Asn Leu Thr Cys Ser Thr Asn Asp Thr Gly

305 310 315 320

Ile Ser Ile Arg Trp Phe Phe Lys Asn Gln Ser Leu Pro Ser Ser Glu

325 330 335

Arg Met Lys Leu Ser Gln Gly Asn Thr Thr Leu Ser Ile Asn Pro Val

340 345 350

Lys Arg Glu Asp Ala Gly Thr Tyr Trp Cys Glu Val Phe Asn Pro Ile

355 360 365

Ser Lys Asn Gln Ser Asp Pro Ile Met Leu Asn Val Asn Tyr Asn Ala

370 375 380

Leu Pro Gln Glu Asn Gly Leu Ser Pro Gly His His His His His

385 390 395 400

His His His His

<210> 164

<211> 657

<212> PRT

<213> Homo sapiens

<400> 164

Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu Gly Lys Glu

1 5 10 15

Val Leu Leu Leu Val His Asn Leu Pro Gln His Leu Phe Gly Tyr Ser

20 25 30

Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gln Ile Ile Gly Tyr

35 40 45

Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser Gly Arg

50 55 60

Glu Ile Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Ile Ile Gln

65 70 75 80

Asn Asp Thr Gly Phe Tyr Thr Leu His Val Ile Lys Ser Asp Leu Val

85 90 95

Asn Glu Glu Ala Thr Gly Gln Phe Arg Val Tyr Pro Glu Leu Pro Lys

100 105 110

Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro Val Glu Asp Lys Asp Ala

115 120 125

Val Ala Phe Thr Cys Glu Pro Glu Thr Gln Asp Ala Thr Tyr Leu Trp

130 135 140

Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln Leu Ser

145 150 155 160

Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn Val Thr Arg Asn Asp Ser

165 170 175

Ala Ser Tyr Lys Cys Glu Thr Gln Asn Pro Val Ser Ala Arg Arg Ser

180 185 190

Asp Ser Val Ile Leu Asn Val Leu Tyr Gly Pro Asp Ala Pro Thr Ile

195 200 205

Ser Pro Leu Asn Thr Ser Tyr Arg Ser Gly Glu Asn Leu Asn Leu Ser

210 215 220

Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Phe Val Asn

225 230 235 240

Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn Ile Thr

245 250 255

Val Asn Asn Ser Gly Ser Tyr Thr Cys Gln Ala His Asn Ser Asp Thr

260 265 270

Gly Leu Asn Arg Thr Thr Val Thr Thr Ile Thr Val Tyr Ala Glu Pro

275 280 285

Pro Lys Pro Phe Ile Thr Ser Asn Asn Ser Asn Pro Val Glu Asp Glu

290 295 300

Asp Ala Val Ala Leu Thr Cys Glu Pro Glu Ile Gln Asn Thr Thr Tyr

305 310 315 320

Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln

325 330 335

Leu Ser Asn Asp Asn Arg Thr Leu Thr Leu Leu Ser Val Thr Arg Asn

340 345 350

Asp Val Gly Pro Tyr Glu Cys Gly Ile Gln Asn Glu Leu Ser Val Asp

355 360 365

His Ser Asp Pro Val Ile Leu Asn Val Leu Tyr Gly Pro Asp Asp Pro

370 375 380

Thr Ile Ser Pro Ser Tyr Thr Tyr Tyr Arg Pro Gly Val Asn Leu Ser

385 390 395 400

Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Leu

405 410 415

Ile Asp Gly Asn Ile Gln Gln His Thr Gln Glu Leu Phe Ile Ser Asn

420 425 430

Ile Thr Glu Lys Asn Ser Gly Leu Tyr Thr Cys Gln Ala Asn Asn Ser

435 440 445

Ala Ser Gly His Ser Arg Thr Thr Val Lys Thr Ile Thr Val Ser Ala

450 455 460

Glu Leu Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro Val Glu

465 470 475 480

Asp Lys Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Ala Gln Asn Thr

485 490 495

Thr Tyr Leu Trp Trp Val Asn Gly Gln Ser Leu Pro Val Ser Pro Arg

500 505 510

Leu Gln Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn Val Thr

515 520 525

Arg Asn Asp Ala Arg Ala Tyr Val Cys Gly Ile Gln Asn Ser Val Ser

530 535 540

Ala Asn Arg Ser Asp Pro Val Thr Leu Asp Val Leu Tyr Gly Pro Asp

545 550 555 560

Thr Pro Ile Ile Ser Pro Pro Asp Ser Ser Tyr Leu Ser Gly Ala Asn

565 570 575

Leu Asn Leu Ser Cys His Ser Ala Ser Asn Pro Ser Pro Gln Tyr Ser

580 585 590

Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu Phe Ile

595 600 605

Ala Lys Ile Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe Val Ser

610 615 620

Asn Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Lys Ser Ile Thr Val

625 630 635 640

Ser Ala Ser Gly Thr Ser Pro Gly Leu Ser Ala His His His His

645 650 655

His

<210> 165

<211> 532

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-1790, hCeacam6-WT-Fc-6xHis

<400> 165

Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu Gly Lys Glu

1 5 10 15

Val Leu Leu Leu Ala His Asn Leu Pro Gln Asn Arg Ile Gly Tyr Ser

20 25 30

Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Ser Leu Ile Val Gly Tyr

35 40 45

Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser Gly Arg

50 55 60

Glu Thr Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Val Thr Gln

65 70 75 80

Asn Asp Thr Gly Phe Tyr Thr Leu Gln Val Ile Lys Ser Asp Leu Val

85 90 95

Asn Glu Glu Ala Thr Gly Gln Phe His Val Tyr Pro Glu Leu Pro Lys

100 105 110

Pro Ser Ile Ser Ser Asn Asn Ser Asn Pro Val Glu Asp Lys Asp Ala

115 120 125

Val Ala Phe Thr Cys Glu Pro Glu Val Gln Asn Thr Thr Tyr Leu Trp

130 135 140

Trp Val Asn Gly Gln Ser Leu Pro Val Ser Pro Arg Leu Gln Leu Ser

145 150 155 160

Asn Gly Asn Met Thr Leu Thr Leu Leu Ser Val Lys Arg Asn Asp Ala

165 170 175

Gly Ser Tyr Glu Cys Glu Ile Gln Asn Pro Ala Ser Ala Asn Arg Ser

180 185 190

Asp Pro Val Thr Leu Asn Val Leu Tyr Gly Pro Asp Gly Pro Thr Ile

195 200 205

Ser Pro Ser Lys Ala Asn Tyr Arg Pro Gly Glu Asn Leu Asn Leu Ser

210 215 220

Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Phe Ile Asn

225 230 235 240

Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn Ile Thr

245 250 255

Val Asn Asn Ser Gly Ser Tyr Met Cys Gln Ala His Asn Ser Ala Thr

260 265 270

Gly Leu Asn Arg Thr Thr Val Thr Met Ile Thr Val Ser Gly Ile Glu

275 280 285

Gly Arg Asp Met Asp Thr Gly Pro Lys Ser Cys Asp Lys Thr His Thr

290 295 300

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

305 310 315 320

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

325 330 335

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

340 345 350

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

355 360 365

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

370 375 380

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

385 390 395 400

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

405 410 415

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

420 425 430

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

435 440 445

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

450 455 460

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

465 470 475 480

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

485 490 495

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

500 505 510

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys His His

515 520 525

His His His His

530

<210> 166

<211> 532

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-1791, hCeacam6-Dom1-MacMul-Xa-Fc-His

<400> 166

Gln Leu Thr Ile Glu Ser Arg Pro Phe Asn Val Ala Glu Gly Lys Glu

1 5 10 15

Val Leu Leu Leu Ala His Asn Leu Pro Gln Asn Leu Ile Gly Phe Asn

20 25 30

Trp Tyr Lys Gly Glu Arg Val Asp Ala Lys Arg Leu Ile Val Ala Tyr

35 40 45

Val Ile Glu Thr Gln Gln Thr Thr Pro Gly Pro Ala His Ser Gly Arg

50 55 60

Glu Thr Val Tyr Ser Asn Ala Ser Leu Leu Ile Gln Asn Val Thr Gln

65 70 75 80

Asn Asp Thr Gly Ser Tyr Thr Leu Gln Val Ile Lys Gly Asp Leu Val

85 90 95

Asn Glu Glu Ala Thr Gly Arg Phe Trp Val Tyr Pro Glu Leu Pro Lys

100 105 110

Pro Ser Ile Ser Ser Asn Asn Ser Asn Pro Val Glu Asp Lys Asp Ala

115 120 125

Val Ala Phe Thr Cys Glu Pro Glu Val Gln Asn Thr Thr Tyr Leu Trp

130 135 140

Trp Val Asn Gly Gln Ser Leu Pro Val Ser Pro Arg Leu Gln Leu Ser

145 150 155 160

Asn Gly Asn Met Thr Leu Thr Leu Leu Ser Val Lys Arg Asn Asp Ala

165 170 175

Gly Ser Tyr Glu Cys Glu Ile Gln Asn Pro Ala Ser Ala Asn Arg Ser

180 185 190

Asp Pro Val Thr Leu Asn Val Leu Tyr Gly Pro Asp Gly Pro Thr Ile

195 200 205

Ser Pro Ser Lys Ala Asn Tyr Arg Pro Gly Glu Asn Leu Asn Leu Ser

210 215 220

Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Phe Ile Asn

225 230 235 240

Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn Ile Thr

245 250 255

Val Asn Asn Ser Gly Ser Tyr Met Cys Gln Ala His Asn Ser Ala Thr

260 265 270

Gly Leu Asn Arg Thr Thr Val Thr Met Ile Thr Val Ser Gly Ile Glu

275 280 285

Gly Arg Asp Met Asp Thr Gly Pro Lys Ser Cys Asp Lys Thr His Thr

290 295 300

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

305 310 315 320

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

325 330 335

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

340 345 350

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

355 360 365

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

370 375 380

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

385 390 395 400

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

405 410 415

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

420 425 430

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

435 440 445

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

450 455 460

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

465 470 475 480

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

485 490 495

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

500 505 510

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys His His

515 520 525

His His His His

530

<210> 167

<211> 532

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-1792, hCeacam6-Dom2-MacMul-Xa-Fc-His

<400> 167

Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu Gly Lys Glu

1 5 10 15

Val Leu Leu Leu Ala His Asn Leu Pro Gln Asn Arg Ile Gly Tyr Ser

20 25 30

Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Ser Leu Ile Val Gly Tyr

35 40 45

Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser Gly Arg

50 55 60

Glu Thr Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Val Thr Gln

65 70 75 80

Asn Asp Thr Gly Phe Tyr Thr Leu Gln Val Ile Lys Ser Asp Leu Val

85 90 95

Asn Glu Glu Ala Thr Gly Gln Phe His Val Tyr Pro Glu Leu Pro Lys

100 105 110

Pro Tyr Ile Thr Ser Asn Asn Ser Asn Pro Val Glu Asp Lys Asp Ala

115 120 125

Val Asp Phe Thr Cys Glu Pro Asp Ile His Ser Thr Thr Tyr Leu Trp

130 135 140

Trp Val Asn Asp Gln Ser Leu Pro Val Ser Pro Arg Leu Gln Leu Ser

145 150 155 160

Asn Gly Asn Arg Thr Leu Thr Leu Leu Ser Val Lys Arg Asn Asp Ala

165 170 175

Gly Ala Tyr Glu Cys Glu Ile Gln Asn Pro Val Ser Ala Asn Leu Ser

180 185 190

Asp Pro Val Ile Leu Asn Val Leu Tyr Gly Pro Asp Gly Pro Thr Ile

195 200 205

Ser Pro Ser Lys Ala Asn Tyr Arg Pro Gly Glu Asn Leu Asn Leu Ser

210 215 220

Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Phe Ile Asn

225 230 235 240

Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn Ile Thr

245 250 255

Val Asn Asn Ser Gly Ser Tyr Met Cys Gln Ala His Asn Ser Ala Thr

260 265 270

Gly Leu Asn Arg Thr Thr Val Thr Met Ile Thr Val Ser Gly Ile Glu

275 280 285

Gly Arg Asp Met Asp Thr Gly Pro Lys Ser Cys Asp Lys Thr His Thr

290 295 300

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

305 310 315 320

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

325 330 335

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

340 345 350

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

355 360 365

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

370 375 380

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

385 390 395 400

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

405 410 415

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

420 425 430

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

435 440 445

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

450 455 460

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

465 470 475 480

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

485 490 495

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

500 505 510

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys His His

515 520 525

His His His His

530

<210> 168

<211> 532

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-1793, hCeacam6-Dom3-MacMul-Xa-Fc-His

<400> 168

Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu Gly Lys Glu

1 5 10 15

Val Leu Leu Leu Ala His Asn Leu Pro Gln Asn Arg Ile Gly Tyr Ser

20 25 30

Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Ser Leu Ile Val Gly Tyr

35 40 45

Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser Gly Arg

50 55 60

Glu Thr Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Val Thr Gln

65 70 75 80

Asn Asp Thr Gly Phe Tyr Thr Leu Gln Val Ile Lys Ser Asp Leu Val

85 90 95

Asn Glu Glu Ala Thr Gly Gln Phe His Val Tyr Pro Glu Leu Pro Lys

100 105 110

Pro Ser Ile Ser Ser Asn Asn Ser Asn Pro Val Glu Asp Lys Asp Ala

115 120 125

Val Ala Phe Thr Cys Glu Pro Glu Val Gln Asn Thr Thr Tyr Leu Trp

130 135 140

Trp Val Asn Gly Gln Ser Leu Pro Val Ser Pro Arg Leu Gln Leu Ser

145 150 155 160

Asn Gly Asn Met Thr Leu Thr Leu Leu Ser Val Lys Arg Asn Asp Ala

165 170 175

Gly Ser Tyr Glu Cys Glu Ile Gln Asn Pro Ala Ser Ala Asn Arg Ser

180 185 190

Asp Pro Val Thr Leu Asn Val Leu Tyr Gly Pro Asp Val Pro Thr Ile

195 200 205

Ser Pro Ser Asn Ser Asn Tyr Arg Pro Gly Glu Asn Leu Asn Leu Ser

210 215 220

Cys His Ala Ala Ser Asn Pro Thr Ala Gln Tyr Ser Trp Phe Val Asn

225 230 235 240

Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn Ile Thr

245 250 255

Val Asn Asn Ser Gly Ser Tyr Met Cys Gln Ala Tyr Asn Ser Ala Thr

260 265 270

Gly Leu Asn Arg Thr Thr Val Met Met Ile Thr Val Ser Gly Ile Glu

275 280 285

Gly Arg Asp Met Asp Thr Gly Pro Lys Ser Cys Asp Lys Thr His Thr

290 295 300

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

305 310 315 320

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

325 330 335

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

340 345 350

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

355 360 365

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

370 375 380

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

385 390 395 400

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

405 410 415

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

420 425 430

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

435 440 445

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

450 455 460

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

465 470 475 480

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

485 490 495

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

500 505 510

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys His His

515 520 525

His His His His

530

<210> 169

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-1794, hCeacam6-Dom1-8xHis (E. coli)

<400> 169

Met Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu Gly Lys

1 5 10 15

Glu Val Leu Leu Leu Ala His Asn Leu Pro Gln Asn Arg Ile Gly Tyr

20 25 30

Ser Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Ser Leu Ile Val Gly

35 40 45

Tyr Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser Gly

50 55 60

Arg Glu Thr Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Val Thr

65 70 75 80

Gln Asn Asp Thr Gly Phe Tyr Thr Leu Gln Val Ile Lys Ser Asp Leu

85 90 95

Val Asn Glu Glu Ala Thr Gly Gln Phe His Val Tyr Pro Gly Ser Gly

100 105 110

Ser His His His His His His His

115 120

<210> 170

<211> 533

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-2443, cynomolgus monkey CEACAM-6-Xa-Fc-His

<400> 170

Gln Leu Thr Ile Glu Ser Arg Pro Phe Asn Val Ala Glu Gly Lys Glu

1 5 10 15

Val Leu Leu Leu Ala His Asn Leu Pro Gln Asn Thr Leu Gly Phe Asn

20 25 30

Trp Tyr Lys Gly Glu Arg Val Asp Ala Lys Arg Leu Ile Val Ala Tyr

35 40 45

Val Ile Gly Thr Gln Gln Thr Thr Pro Gly Pro Ala His Ser Gly Arg

50 55 60

Glu Met Ile Tyr Ser Asn Ala Ser Leu Leu Ile Gln Asn Val Thr Gln

65 70 75 80

Asn Asp Thr Gly Ser Tyr Thr Leu Gln Ala Ile Lys Glu Asp Leu Val

85 90 95

Thr Glu Glu Ala Thr Gly Arg Phe Trp Val Tyr Pro Glu Leu Pro Lys

100 105 110

Pro Tyr Ile Thr Ser Asn Asn Ser Asn Pro Val Glu Asp Lys Asp Ala

115 120 125

Val Asp Phe Thr Cys Glu Pro Asp Ile His Ser Thr Thr Tyr Leu Trp

130 135 140

Trp Val Asn Asp Gln Ser Leu Pro Val Ser Pro Arg Leu Gln Leu Ser

145 150 155 160

Asn Gly Asn Arg Thr Leu Thr Leu Leu Ser Val Lys Arg Asn Asp Ala

165 170 175

Gly Ala Tyr Glu Cys Glu Ile Gln Asn Pro Val Ser Ala Asn Leu Ser

180 185 190

Asp Pro Val Ile Leu Asn Val Leu Tyr Gly Pro Asp Val Pro Thr Ile

195 200 205

Ser Pro Ser Asn Ser Asn Tyr Arg Pro Gly Glu Asn Leu Asn Leu Ser

210 215 220

Cys His Ala Ala Ser Asn Pro Thr Ala Gln Tyr Ser Trp Phe Val Asn

225 230 235 240

Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn Ile Thr

245 250 255

Val Asn Asn Ser Gly Ser Tyr Met Cys Gln Ala Tyr Asn Ser Ala Thr

260 265 270

Gly Leu Asn Arg Thr Thr Val Met Met Ile Thr Val Ser Gly Ser Ile

275 280 285

Glu Gly Arg Asp Met Asp Thr Gly Pro Lys Ser Cys Asp Lys Thr His

290 295 300

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

305 310 315 320

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

325 330 335

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

340 345 350

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

355 360 365

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

370 375 380

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

385 390 395 400

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

405 410 415

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

420 425 430

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

435 440 445

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

450 455 460

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

465 470 475 480

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

485 490 495

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

500 505 510

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys His

515 520 525

His His His His His

530

<210> 171

<211> 356

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-2452, cynomolgus monkey Ceacam6-Dom1-Xa-Fc-His

<400> 171

Gln Leu Thr Ile Glu Ser Arg Pro Phe Asn Val Ala Glu Gly Lys Glu

1 5 10 15

Val Leu Leu Leu Ala His Asn Leu Pro Gln Asn Thr Leu Gly Phe Asn

20 25 30

Trp Tyr Lys Gly Glu Arg Val Asp Ala Lys Arg Leu Ile Val Ala Tyr

35 40 45

Val Ile Gly Thr Gln Gln Thr Thr Pro Gly Pro Ala His Ser Gly Arg

50 55 60

Glu Met Ile Tyr Ser Asn Ala Ser Leu Leu Ile Gln Asn Val Thr Gln

65 70 75 80

Asn Asp Thr Gly Ser Tyr Thr Leu Gln Ala Ile Lys Glu Asp Leu Val

85 90 95

Thr Glu Glu Ala Thr Gly Arg Phe Trp Val Tyr Pro Glu Leu Ile Glu

100 105 110

Gly Arg Asp Met Asp Thr Gly Pro Lys Ser Cys Asp Lys Thr His Thr

115 120 125

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

130 135 140

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

145 150 155 160

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

165 170 175

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

180 185 190

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

195 200 205

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

210 215 220

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

225 230 235 240

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

245 250 255

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

260 265 270

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

275 280 285

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

290 295 300

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

305 310 315 320

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

325 330 335

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys His His

340 345 350

His His His His

355

<210> 172

<211> 127

<212> PRT

<213> Homo sapiens

<400> 172

Lys Leu Thr Ile Glu Ser Met Pro Leu Ser Val Ala Glu Gly Lys Glu

1 5 10 15

Val Leu Leu Leu Val His Asn Leu Pro Gln His Leu Phe Gly Tyr Ser

20 25 30

Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Ser Leu Ile Val Gly Tyr

35 40 45

Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Ala Ala Tyr Ser Gly Arg

50 55 60

Glu Thr Ile Tyr Thr Asn Ala Ser Leu Leu Ile Gln Asn Val Thr Gln

65 70 75 80

Asn Asp Ile Gly Phe Tyr Thr Leu Gln Val Ile Lys Ser Asp Leu Val

85 90 95

Asn Glu Glu Ala Thr Gly Gln Phe His Val Tyr Gln Glu Asn Ala Pro

100 105 110

Gly Leu Pro Val Gly Ala Val Ala Gly His His His His His

115 120 125

<210> 173

<211> 526

<212> PRT

<213> Homo sapiens

<400> 173

Met Gly His Leu Ser Ala Pro Leu His Arg Val Arg Val Pro Trp Gln

1 5 10 15

Gly Leu Leu Leu Thr Ala Ser Leu Leu Thr Phe Trp Asn Pro Pro Thr

20 25 30

Thr Ala Gln Leu Thr Thr Glu Ser Met Pro Phe Asn Val Ala Glu Gly

35 40 45

Lys Glu Val Leu Leu Leu Val His Asn Leu Pro Gln Gln Leu Phe Gly

50 55 60

Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gln Ile Val

65 70 75 80

Gly Tyr Ala Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Asn Ser

85 90 95

Gly Arg Glu Thr Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Val

100 105 110

Thr Gln Asn Asp Thr Gly Phe Tyr Thr Leu Gln Val Ile Lys Ser Asp

115 120 125

Leu Val Asn Glu Glu Ala Thr Gly Gln Phe His Val Tyr Pro Glu Leu

130 135 140

Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Asn Pro Val Glu Asp Lys

145 150 155 160

Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Thr Gln Asp Thr Thr Tyr

165 170 175

Leu Trp Trp Ile Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln

180 185 190

Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Leu Ser Val Thr Arg Asn

195 200 205

Asp Thr Gly Pro Tyr Glu Cys Glu Ile Gln Asn Pro Val Ser Ala Asn

210 215 220

Arg Ser Asp Pro Val Thr Leu Asn Val Thr Tyr Gly Pro Asp Thr Pro

225 230 235 240

Thr Ile Ser Pro Ser Asp Thr Tyr Tyr Arg Pro Gly Ala Asn Leu Ser

245 250 255

Leu Ser Cys Tyr Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Leu

260 265 270

Ile Asn Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn

275 280 285

Ile Thr Val Asn Asn Ser Gly Ser Tyr Thr Cys His Ala Asn Asn Ser

290 295 300

Val Thr Gly Cys Asn Arg Thr Thr Val Lys Thr Ile Ile Val Thr Glu

305 310 315 320

Leu Ser Pro Val Val Ala Lys Pro Gln Ile Lys Ala Ser Lys Thr Thr

325 330 335

Val Thr Gly Asp Lys Asp Ser Val Asn Leu Thr Cys Ser Thr Asn Asp

340 345 350

Thr Gly Ile Ser Ile Arg Trp Phe Phe Lys Asn Gln Ser Leu Pro Ser

355 360 365

Ser Glu Arg Met Lys Leu Ser Gln Gly Asn Thr Thr Leu Ser Ile Asn

370 375 380

Pro Val Lys Arg Glu Asp Ala Gly Thr Tyr Trp Cys Glu Val Phe Asn

385 390 395 400

Pro Ile Ser Lys Asn Gln Ser Asp Pro Ile Met Leu Asn Val Asn Tyr

405 410 415

Asn Ala Leu Pro Gln Glu Asn Gly Leu Ser Pro Gly Ala Ile Ala Gly

420 425 430

Ile Val Ile Gly Val Val Ala Leu Val Ala Leu Ile Ala Val Ala Leu

435 440 445

Ala Cys Phe Leu His Phe Gly Lys Thr Gly Arg Ala Ser Asp Gln Arg

450 455 460

Asp Leu Thr Glu His Lys Pro Ser Val Ser Asn His Thr Gln Asp His

465 470 475 480

Ser Asn Asp Pro Pro Asn Lys Met Asn Glu Val Thr Tyr Ser Thr Leu

485 490 495

Asn Phe Glu Ala Gln Gln Pro Thr Gln Pro Thr Ser Ala Ser Pro Ser

500 505 510

Leu Thr Ala Thr Glu Ile Ile Tyr Ser Glu Val Lys Lys Gln

515 520 525

<210> 174

<211> 300

<212> PRT

<213> Homo sapiens

<400> 174

Met Glu Ile Pro Met Gly Thr Gln Gly Cys Phe Ser Lys Ser Leu Leu

1 5 10 15

Leu Ser Ala Ser Ile Leu Val Leu Trp Met Leu Gln Gly Ser Gln Ala

20 25 30

Ala Leu Tyr Ile Gln Lys Ile Pro Glu Gln Pro Gln Lys Asn Gln Asp

35 40 45

Leu Leu Leu Ser Val Gln Gly Val Pro Asp Thr Phe Gln Asp Phe Asn

50 55 60

Trp Tyr Leu Gly Glu Glu Thr Tyr Tyr Gly Gly Thr Arg Leu Phe Thr Tyr

65 70 75 80

Ile Pro Gly Ile Gln Arg Pro Gln Arg Asp Gly Ser Ala Met Gly Gln

85 90 95

Arg Asp Ile Val Gly Phe Pro Asn Gly Ser Met Leu Leu Arg Arg Ala

100 105 110

Gln Pro Thr Asp Ser Gly Thr Tyr Gln Val Ala Ile Thr Ile Asn Ser

115 120 125

Glu Trp Thr Met Lys Ala Lys Thr Glu Val Gln Val Ala Glu Lys Asn

130 135 140

Lys Glu Leu Pro Ser Thr His Leu Pro Thr Asn Ala Gly Ile Leu Ala

145 150 155 160

Ala Thr Ile Ile Gly Ser Leu Ala Ala Gly Ala Leu Leu Ile Ser Cys

165 170 175

Ile Ala Tyr Leu Leu Val Thr Arg Asn Trp Arg Gly Gln Ser His Arg

180 185 190

Leu Pro Ala Pro Arg Gly Gln Gly Ser Leu Ser Ile Leu Cys Ser Ala

195 200 205

Val Ser Pro Val Pro Ser Val Thr Pro Ser Thr Trp Met Ala Thr Thr

210 215 220

Glu Lys Pro Glu Leu Gly Pro Ala His Asp Ala Gly Asp Asn Asn Ile

225 230 235 240

Tyr Glu Val Met Pro Ser Pro Val Leu Leu Val Ser Pro Ile Ser Asp

245 250 255

Thr Arg Ser Ile Asn Pro Ala Arg Pro Leu Pro Thr Pro Pro His Leu

260 265 270

Gln Ala Glu Pro Glu Asn His Gln Tyr Gln Gln Asp Leu Leu Asn Pro

275 280 285

Asp Pro Ala Pro Tyr Cys Gln Leu Val Pro Thr Ser

290 295 300

<210> 175

<211> 252

<212> PRT

<213> Homo sapiens

<400> 175

Met Gly Pro Pro Ser Ala Ser Pro His Arg Glu Cys Ile Pro Trp Gln

1 5 10 15

Gly Leu Leu Leu Thr Ala Ser Leu Leu Asn Phe Trp Asn Pro Pro Thr

20 25 30

Thr Ala Lys Leu Thr Ile Glu Ser Met Pro Leu Ser Val Ala Glu Gly

35 40 45

Lys Glu Val Leu Leu Leu Val His Asn Leu Pro Gln His Leu Phe Gly

50 55 60

Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Ser Leu Ile Val

65 70 75 80

Gly Tyr Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Ala Ala Tyr Ser

85 90 95

Gly Arg Glu Thr Ile Tyr Thr Asn Ala Ser Leu Leu Ile Gln Asn Val

100 105 110

Thr Gln Asn Asp Ile Gly Phe Tyr Thr Leu Gln Val Ile Lys Ser Asp

115 120 125

Leu Val Asn Glu Glu Ala Thr Gly Gln Phe His Val Tyr Gln Glu Asn

130 135 140

Ala Pro Gly Leu Pro Val Gly Ala Val Ala Gly Ile Val Thr Gly Val

145 150 155 160

Leu Val Gly Val Ala Leu Val Ala Ala Leu Val Cys Phe Leu Leu Leu

165 170 175

Ala Lys Thr Gly Arg Thr Ser Ile Gln Arg Asp Leu Lys Glu Gln Gln

180 185 190

Pro Gln Ala Leu Ala Pro Gly Arg Gly Pro Ser His Ser Ser Ala Phe

195 200 205

Ser Met Ser Pro Leu Ser Thr Ala Gln Ala Pro Leu Pro Asn Pro Arg

210 215 220

Thr Ala Ala Ser Ile Tyr Glu Glu Leu Leu Lys His Asp Thr Asn Ile

225 230 235 240

Tyr Cys Arg Met Asp His Lys Ala Glu Val Ala Ser

245 250

<210> 176

<211> 702

<212> PRT

<213> Homo sapiens

<400> 176

Met Glu Ser Pro Ser Ala Pro Pro His Arg Trp Cys Ile Pro Trp Gln

1 5 10 15

Arg Leu Leu Leu Thr Ala Ser Leu Leu Thr Phe Trp Asn Pro Pro Thr

20 25 30

Thr Ala Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu Gly

35 40 45

Lys Glu Val Leu Leu Leu Val His Asn Leu Pro Gln His Leu Phe Gly

50 55 60

Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gln Ile Ile

65 70 75 80

Gly Tyr Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser

85 90 95

Gly Arg Glu Ile Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Ile

100 105 110

Ile Gln Asn Asp Thr Gly Phe Tyr Thr Leu His Val Ile Lys Ser Asp

115 120 125

Leu Val Asn Glu Glu Ala Thr Gly Gln Phe Arg Val Tyr Pro Glu Leu

130 135 140

Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro Val Glu Asp Lys

145 150 155 160

Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Thr Gln Asp Ala Thr Tyr

165 170 175

Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln

180 185 190

Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn Val Thr Arg Asn

195 200 205

Asp Thr Ala Ser Tyr Lys Cys Glu Thr Gln Asn Pro Val Ser Ala Arg

210 215 220

Arg Ser Asp Ser Val Ile Leu Asn Val Leu Tyr Gly Pro Asp Ala Pro

225 230 235 240

Thr Ile Ser Pro Leu Asn Thr Ser Tyr Arg Ser Gly Glu Asn Leu Asn

245 250 255

Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Phe

260 265 270

Val Asn Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn

275 280 285

Ile Thr Val Asn Asn Ser Gly Ser Tyr Thr Cys Gln Ala His Asn Ser

290 295 300

Asp Thr Gly Leu Asn Arg Thr Thr Val Thr Thr Ile Thr Val Tyr Ala

305 310 315 320

Glu Pro Pro Lys Pro Phe Ile Thr Ser Asn Asn Ser Asn Pro Val Glu

325 330 335

Asp Glu Asp Ala Val Ala Leu Thr Cys Glu Pro Glu Ile Gln Asn Thr

340 345 350

Thr Tyr Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg

355 360 365

Leu Gln Leu Ser Asn Asp Asn Arg Thr Leu Thr Leu Leu Ser Val Thr

370 375 380

Arg Asn Asp Val Gly Pro Tyr Glu Cys Gly Ile Gln Asn Lys Leu Ser

385 390 395 400

Val Asp His Ser Asp Pro Val Ile Leu Asn Val Leu Tyr Gly Pro Asp

405 410 415

Asp Pro Thr Ile Ser Pro Ser Tyr Thr Tyr Tyr Arg Pro Gly Val Asn

420 425 430

Leu Ser Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser

435 440 445

Trp Leu Ile Asp Gly Asn Ile Gln Gln His Thr Gln Glu Leu Phe Ile

450 455 460

Ser Asn Ile Thr Glu Lys Asn Ser Gly Leu Tyr Thr Cys Gln Ala Asn

465 470 475 480

Asn Ser Ala Ser Gly His Ser Arg Thr Thr Val Lys Thr Ile Thr Val

485 490 495

Ser Ala Glu Leu Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro

500 505 510

Val Glu Asp Lys Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Ala Gln

515 520 525

Asn Thr Thr Tyr Leu Trp Trp Val Asn Gly Gln Ser Leu Pro Val Ser

530 535 540

Pro Arg Leu Gln Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn

545 550 555 560

Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Cys Gly Ile Gln Asn Ser

565 570 575

Val Ser Ala Asn Arg Ser Asp Pro Val Thr Leu Asp Val Leu Tyr Gly

580 585 590

Pro Asp Thr Pro Ile Ile Ser Pro Pro Asp Ser Ser Tyr Leu Ser Gly

595 600 605

Ala Asn Leu Asn Leu Ser Cys His Ser Ala Ser Asn Pro Ser Pro Gln

610 615 620

Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu

625 630 635 640

Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe

645 650 655

Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Lys Ser Ile

660 665 670

Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu Ser Ala Gly Ala Thr

675 680 685

Val Gly Ile Met Ile Gly Val Leu Val Gly Val Ala Leu Ile

690 695 700

<210> 177

<211> 344

<212> PRT

<213> Macaca fascicularis (Cynomolgus monkey)

<400> 177

Met Gly Pro Pro Ser Ala Pro Pro Cys Arg Ile Cys Val Pro Trp Lys

1 5 10 15

Glu Val Leu Leu Thr Ala Ser Leu Leu Thr Phe Trp Ser Pro Pro Thr

20 25 30

Thr Ala Gln Leu Thr Ile Glu Ser Arg Pro Phe Asn Val Ala Glu Gly

35 40 45

Lys Glu Val Leu Leu Leu Ala His Asn Leu Pro Gln Asn Thr Leu Gly

50 55 60

Phe Asn Trp Tyr Lys Gly Glu Arg Val Asp Ala Lys Arg Leu Ile Val

65 70 75 80

Ala Tyr Val Ile Gly Thr Gln Gln Thr Thr Pro Gly Pro Ala His Ser

85 90 95

Gly Arg Glu Met Ile Tyr Ser Asn Ala Ser Leu Leu Ile Gln Asn Val

100 105 110

Thr Gln Asn Asp Thr Gly Ser Tyr Thr Leu Gln Ala Ile Lys Glu Asp

115 120 125

Leu Val Thr Glu Glu Ala Thr Gly Arg Phe Trp Val Tyr Pro Glu Leu

130 135 140

Pro Lys Pro Tyr Ile Thr Ser Asn Asn Ser Asn Pro Val Glu Asp Lys

145 150 155 160

Asp Ala Val Asp Phe Thr Cys Glu Pro Asp Ile His Ser Thr Thr Tyr

165 170 175

Leu Trp Trp Val Asn Asp Gln Ser Leu Pro Val Ser Pro Arg Leu Gln

180 185 190

Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Leu Ser Val Lys Arg Asn

195 200 205

Asp Ala Gly Ala Tyr Glu Cys Glu Ile Gln Asn Pro Val Ser Ala Asn

210 215 220

Leu Ser Asp Pro Val Ile Leu Asn Val Leu Tyr Gly Pro Asp Val Pro

225 230 235 240

Thr Ile Ser Pro Ser Asn Ser Asn Tyr Arg Pro Gly Glu Asn Leu Asn

245 250 255

Leu Ser Cys His Ala Ala Ser Asn Pro Thr Ala Gln Tyr Ser Trp Phe

260 265 270

Val Asn Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn

275 280 285

Ile Thr Val Asn Asn Ser Gly Ser Tyr Met Cys Gln Ala Tyr Asn Ser

290 295 300

Ala Thr Gly Leu Asn Arg Thr Thr Val Met Met Ile Thr Val Ser Gly

305 310 315 320

Ser Ala Pro Gly Leu Ser Ala Val Ala Thr Val Gly Ile Met Ile Gly

325 330 335

Val Leu Ala Arg Val Ala Leu Ile

340

<210> 178

<211> 349

<212> PRT

<213> Homo sapiens

<400> 178

Met Gly Pro Ile Ser Ala Pro Ser Cys Arg Trp Arg Ile Pro Trp Gln

1 5 10 15

Gly Leu Leu Leu Thr Ala Ser Leu Phe Thr Phe Trp Asn Pro Pro Thr

20 25 30

Thr Ala Gln Leu Thr Ile Glu Ala Val Pro Ser Asn Ala Ala Glu Gly

35 40 45

Lys Glu Val Leu Leu Leu Val His Asn Leu Pro Gln Asp Pro Arg Gly

50 55 60

Tyr Asn Trp Tyr Lys Gly Glu Thr Val Asp Ala Asn Arg Arg Ile Ile

65 70 75 80

Gly Tyr Val Ile Ser Asn Gln Gln Ile Thr Pro Gly Pro Ala Tyr Ser

85 90 95

Asn Arg Glu Thr Ile Tyr Pro Asn Ala Ser Leu Leu Met Arg Asn Val

100 105 110

Thr Arg Asn Asp Thr Gly Ser Tyr Thr Leu Gln Val Ile Lys Leu Asn

115 120 125

Leu Met Ser Glu Glu Val Thr Gly Gln Phe Ser Val His Pro Glu Thr

130 135 140

Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Asn Pro Val Glu Asp Lys

145 150 155 160

Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Thr Gln Asn Thr Thr Tyr

165 170 175

Leu Trp Trp Val Asn Gly Gln Ser Leu Pro Val Ser Pro Arg Leu Gln

180 185 190

Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Leu Ser Val Thr Arg Asn

195 200 205

Asp Val Gly Pro Tyr Glu Cys Glu Ile Gln Asn Pro Ala Ser Ala Asn

210 215 220

Phe Ser Asp Pro Val Thr Leu Asn Val Leu Tyr Gly Pro Asp Ala Pro

225 230 235 240

Thr Ile Ser Pro Ser Asp Thr Tyr Tyr His Ala Gly Val Asn Leu Asn

245 250 255

Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ser Gln Tyr Ser Trp Ser

260 265 270

Val Asn Gly Thr Phe Gln Gln Tyr Thr Gln Lys Leu Phe Ile Pro Asn

275 280 285

Ile Thr Thr Lys Asn Ser Gly Ser Tyr Ala Cys His Thr Thr Asn Ser

290 295 300

Ala Thr Gly Arg Asn Arg Thr Thr Val Arg Met Ile Thr Val Ser Asp

305 310 315 320

Ala Leu Val Gln Gly Ser Ser Pro Gly Leu Ser Ala Arg Ala Thr Val

325 330 335

Ser Ile Met Ile Gly Val Leu Ala Arg Val Ala Leu Ile

340 345

<210> 179

<211> 344

<212> PRT

<213> Homo sapiens

<400> 179

Met Gly Pro Pro Ser Ala Pro Pro Cys Arg Leu His Val Pro Trp Lys

1 5 10 15

Glu Val Leu Leu Thr Ala Ser Leu Leu Thr Phe Trp Asn Pro Pro Thr

20 25 30

Thr Ala Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu Gly

35 40 45

Lys Glu Val Leu Leu Leu Ala His Asn Leu Pro Gln Asn Arg Ile Gly

50 55 60

Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Ser Leu Ile Val

65 70 75 80

Gly Tyr Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser

85 90 95

Gly Arg Glu Thr Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Val

100 105 110

Thr Gln Asn Asp Thr Gly Phe Tyr Thr Leu Gln Val Ile Lys Ser Asp

115 120 125

Leu Val Asn Glu Glu Ala Thr Gly Gln Phe His Val Tyr Pro Glu Leu

130 135 140

Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Asn Pro Val Glu Asp Lys

145 150 155 160

Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Val Gln Asn Thr Thr Tyr

165 170 175

Leu Trp Trp Val Asn Gly Gln Ser Leu Pro Val Ser Pro Arg Leu Gln

180 185 190

Leu Ser Asn Gly Asn Met Thr Leu Thr Leu Leu Ser Val Lys Arg Asn

195 200 205

Asp Ala Gly Ser Tyr Glu Cys Glu Ile Gln Asn Pro Ala Ser Ala Asn

210 215 220

Arg Ser Asp Pro Val Thr Leu Asn Val Leu Tyr Gly Pro Asp Gly Pro

225 230 235 240

Thr Ile Ser Pro Ser Lys Ala Asn Tyr Arg Pro Gly Glu Asn Leu Asn

245 250 255

Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Phe

260 265 270

Ile Asn Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn

275 280 285

Ile Thr Val Asn Asn Ser Gly Ser Tyr Met Cys Gln Ala His Asn Ser

290 295 300

Ala Thr Gly Leu Asn Arg Thr Thr Val Thr Met Ile Thr Val Ser Gly

305 310 315 320

Ser Ala Pro Val Leu Ser Ala Val Ala Thr Val Gly Ile Thr Ile Gly

325 330 335

Val Leu Ala Arg Val Ala Leu Ile

340

<210> 180

<211> 123

<212> PRT

<213> Artificial sequence

<220>

<223> Cynomolgus monkey CEACAM6-domain 1-His

<400> 180

Met Gln Leu Thr Ile Glu Ser Arg Pro Phe Asn Val Ala Glu Gly Lys

1 5 10 15

Glu Val Leu Leu Leu Ala His Asn Leu Pro Gln Asn Thr Leu Gly Phe

20 25 30

Asn Trp Tyr Lys Gly Glu Arg Val Asp Ala Lys Arg Leu Ile Val Ala

35 40 45

Tyr Val Ile Gly Thr Gln Gln Thr Thr Pro Gly Pro Ala His Ser Gly

50 55 60

Arg Glu Met Ile Tyr Ser Asn Ala Ser Leu Leu Ile Gln Asn Val Thr

65 70 75 80

Gln Asn Asp Thr Gly Ser Tyr Thr Leu Gln Ala Ile Lys Glu Asp Leu

85 90 95

Val Thr Glu Glu Ala Thr Gly Arg Phe Trp Val Tyr Pro Glu Leu Gly

100 105 110

Ser Gly Ser His His His His His His

115 120

<210> 181

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-5468 - Heavy Chain

<400> 181

Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Tyr

20 25 30

Gly Ile Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Ser Thr Ser

50 55 60

Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Ser Leu Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 182

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-5468 - Light Chain

<400> 182

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 183

<211> 225

<212> PRT

<213> Artificial sequence

<220>

<223> APP-1574 - Heavy Chain (Fab)

<400> 183

Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Tyr

20 25 30

Gly Ile Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asn Asp Asn Lys Tyr Tyr Ser Thr Ser

50 55 60

Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Ile Ser Leu Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His

225

<210> 184

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> APP-1574 - Light chain (Fab)

<400> 184

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 185

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-8697, hCEACAM6-Domain 1-His - I30L

<400> 185

Met Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu Gly Lys

1 5 10 15

Glu Val Leu Leu Leu Ala His Asn Leu Pro Gln Asn Arg Leu Gly Tyr

20 25 30

Ser Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Ser Leu Ile Val Gly

35 40 45

Tyr Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser Gly

50 55 60

Arg Glu Thr Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Val Thr

65 70 75 80

Gln Asn Asp Thr Gly Phe Tyr Thr Leu Gln Val Ile Lys Ser Asp Leu

85 90 95

Val Asn Glu Glu Ala Thr Gly Gln Phe His Val Tyr Pro Gly Ser Gly

100 105 110

Ser His His His His His His His

115 120

<210> 186

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> TPP-8698, hCEACAM6-Domain 1-His - I30F

<400> 186

Met Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu Gly Lys

1 5 10 15

Glu Val Leu Leu Leu Ala His Asn Leu Pro Gln Asn Arg Phe Gly Tyr

20 25 30

Ser Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Ser Leu Ile Val Gly

35 40 45

Tyr Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser Gly

50 55 60

Arg Glu Thr Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Val Thr

65 70 75 80

Gln Asn Asp Thr Gly Phe Tyr Thr Leu Gln Val Ile Lys Ser Asp Leu

85 90 95

Val Asn Glu Glu Ala Thr Gly Gln Phe His Val Tyr Pro Gly Ser Gly

100 105 110

Ser His His His His His His His

115 120

Claims (24)

1. Antibodies or antigen-binding fragments thereof that specifically bind to human CEACAM6 and cynomolgus monkey (Macaca fascicularis) CEACAM6, comprising... i. Containing heavy chain antigen-binding regions of H-CDR1 (SEQ ID NO:48), H-CDR2 (SEQ ID NO:49), and H-CDR3 (SEQ ID NO:50) and light chain antigen-binding regions of L-CDR1 (SEQ ID NO:52), L-CDR2 (SEQ ID NO:53), and L-CDR3 (SEQ ID NO:54), or ii. Contains heavy chain antigen-binding regions of H-CDR1 shown in SEQ ID NO:106, H-CDR2 shown in SEQ ID NO:107, and H-CDR3 shown in SEQ ID NO:108, and light chain antigen-binding regions of L-CDR1 shown in SEQ ID NO:110, L-CDR2 shown in SEQ ID NO:111, and L-CDR3 shown in SEQ ID NO:112, or iii. Contains heavy chain antigen-binding regions of H-CDR1 shown in SEQ ID NO:4, H-CDR2 shown in SEQ ID NO:5, and H-CDR3 shown in SEQ ID NO:6, and light chain antigen-binding regions of L-CDR1 shown in SEQ ID NO:8, L-CDR2 shown in SEQ ID NO:9, and L-CDR3 shown in SEQ ID NO:10, or iv. Containing heavy chain antigen-binding regions of H-CDR1 shown in SEQ ID NO:34, H-CDR2 shown in SEQ ID NO:35, and H-CDR3 shown in SEQ ID NO:36, and light chain antigen-binding regions of L-CDR1 shown in SEQ ID NO:38, L-CDR2 shown in SEQ ID NO:39, and L-CDR3 shown in SEQ ID NO:40, or v. Containing heavy chain antigen-binding regions of H-CDR1 (SEQ ID NO: 120), H-CDR2 (SEQ ID NO: 121), and H-CDR3 (SEQ ID NO: 122), and light chain antigen-binding regions of L-CDR1 (SEQ ID NO: 124), L-CDR2 (SEQ ID NO: 125), and L-CDR3 (SEQ ID NO: 126), or vi. Contains heavy chain antigen-binding regions of H-CDR1 shown in SEQ ID NO:24, H-CDR2 shown in SEQ ID NO:25, and H-CDR3 shown in SEQ ID NO:26, and light chain antigen-binding regions of L-CDR1 shown in SEQ ID NO:28, L-CDR2 shown in SEQ ID NO:29, and L-CDR3 shown in SEQ ID NO:30, or vii. Contains heavy chain antigen-binding regions of H-CDR1 shown in SEQ ID NO:76, H-CDR2 shown in SEQ ID NO:77, and H-CDR3 shown in SEQ ID NO:78, and light chain antigen-binding regions of L-CDR1 shown in SEQ ID NO:80, L-CDR2 shown in SEQ ID NO:81, and L-CDR3 shown in SEQ ID NO:82, or viii. A heavy chain antigen-binding region containing H-CDR1 as shown in SEQ ID NO:134, H-CDR2 as shown in SEQ ID NO:135, and H-CDR3 as shown in SEQ ID NO:136, and a light chain antigen-binding region containing L-CDR1 as shown in SEQ ID NO:138, L-CDR2 as shown in SEQ ID NO:139, and L-CDR3 as shown in SEQ ID NO:140, or ix. A heavy chain antigen-binding region containing H-CDR1 as shown in SEQ ID NO:148, H-CDR2 as shown in SEQ ID NO:149, and H-CDR3 as shown in SEQ ID NO:150, and a light chain antigen-binding region containing L-CDR1 as shown in SEQ ID NO:152, L-CDR2 as shown in SEQ ID NO:153, and L-CDR3 as shown in SEQ ID NO:154, or x. Contains heavy chain antigen-binding regions of H-CDR1 shown in SEQ ID NO:14, H-CDR2 shown in SEQ ID NO:15, and H-CDR3 shown in SEQ ID NO:16, and light chain antigen-binding regions of L-CDR1 shown in SEQ ID NO:18, L-CDR2 shown in SEQ ID NO:19, and L-CDR3 shown in SEQ ID NO:20, or xi. Contains heavy chain antigen-binding regions of H-CDR1 shown in SEQ ID NO:62, H-CDR2 shown in SEQ ID NO:63, and H-CDR3 shown in SEQ ID NO:64, and light chain antigen-binding regions of L-CDR1 shown in SEQ ID NO:66, L-CDR2 shown in SEQ ID NO:67, and L-CDR3 shown in SEQ ID NO:68, or xii. Contains heavy chain antigen-binding regions of H-CDR1 shown in SEQ ID NO:92, H-CDR2 shown in SEQ ID NO:93, and H-CDR3 shown in SEQ ID NO:94, and light chain antigen-binding regions of L-CDR1 shown in SEQ ID NO:96, L-CDR2 shown in SEQ ID NO:97, and L-CDR3 shown in SEQ ID NO:98. 2. The antibody or antigen-binding fragment thereof according to claim 1, comprising: i. Variable heavy chain sequences as shown in SEQ ID NO:47 and variable light chain sequences as shown in SEQ ID NO:51, or ii. A variable heavy chain sequence as shown in SEQ ID NO:105 and a variable light chain sequence as shown in SEQ ID NO:109, or iii. A variable heavy chain sequence as shown in SEQ ID NO:3 and a variable light chain sequence as shown in SEQ ID NO:7, or iv. Variable heavy chain sequences as shown in SEQ ID NO:33 and variable light chain sequences as shown in SEQ ID NO:37, or v. Variable heavy chain sequences as shown in SEQ ID NO:119 and variable light chain sequences as shown in SEQ ID NO:123, or vi. Variable heavy chain sequences as shown in SEQ ID NO:23 and variable light chain sequences as shown in SEQ ID NO:27, or vii. Variable heavy chain sequences as shown in SEQ ID NO:75 and variable light chain sequences as shown in SEQ ID NO:79, or viii. Variable heavy chain sequences as shown in SEQ ID NO:133 and variable light chain sequences as shown in SEQ ID NO:137, or ix. Variable heavy chain sequences as shown in SEQ ID NO:147 and variable light chain sequences as shown in SEQ ID NO:151, or x. Variable heavy chain sequences as shown in SEQ ID NO:13 and variable light chain sequences as shown in SEQ ID NO:17, or xi. Variable heavy chain sequences as shown in SEQ ID NO:61 and variable light chain sequences as shown in SEQ ID NO:65, or xii. Variable heavy chain sequences as shown in SEQ ID NO:91 and variable light chain sequences as shown in SEQ ID NO:95. 3. The antibody or its antigen-binding fragment according to claim 1 or 2, wherein the antibody is an IgG antibody. 4. The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising: i. The heavy chain region corresponding to SEQ ID NO:57 and the light chain region corresponding to SEQ ID NO:58, or ii. The heavy chain region corresponding to SEQ ID NO:115 and the light chain region corresponding to SEQ ID NO:116, or iii. The heavy chain region corresponding to SEQ ID NO:43 and the light chain region corresponding to SEQ ID NO:44, or iv. The heavy chain region corresponding to SEQ ID NO:129 and the light chain region corresponding to SEQ ID NO:130, or v. The heavy chain region corresponding to SEQ ID NO:85 and the light chain region corresponding to SEQ ID NO:86, or vi. The heavy chain region corresponding to SEQ ID NO:143 and the light chain region corresponding to SEQ ID NO:144, or vii. The heavy chain region corresponding to SEQ ID NO:157 and the light chain region corresponding to SEQ ID NO:158, or viii. The heavy chain region corresponding to SEQ ID NO:71 and the light chain region corresponding to SEQ ID NO:72, or ix. The heavy chain region corresponding to SEQ ID NO:101 and the light chain region corresponding to SEQ ID NO:102. 5. The antibody or antigen-binding fragment thereof according to claim 3, comprising: i. The heavy chain region corresponding to SEQ ID NO:57 and the light chain region corresponding to SEQ ID NO:58, or ii. The heavy chain region corresponding to SEQ ID NO:115 and the light chain region corresponding to SEQ ID NO:116, or iii. The heavy chain region corresponding to SEQ ID NO:43 and the light chain region corresponding to SEQ ID NO:44, or iv. The heavy chain region corresponding to SEQ ID NO:129 and the light chain region corresponding to SEQ ID NO:130, or v. The heavy chain region corresponding to SEQ ID NO:85 and the light chain region corresponding to SEQ ID NO:86, or vi. The heavy chain region corresponding to SEQ ID NO:143 and the light chain region corresponding to SEQ ID NO:144, or vii. The heavy chain region corresponding to SEQ ID NO:157 and the light chain region corresponding to SEQ ID NO:158, or viii. The heavy chain region corresponding to SEQ ID NO:71 and the light chain region corresponding to SEQ ID NO:72, or ix. The heavy chain region corresponding to SEQ ID NO:101 and the light chain region corresponding to SEQ ID NO:102. 6. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antigen-binding fragment is scFv, Fab, Fab' fragment or F(ab')2 fragment. 7. The antibody or antigen-binding fragment thereof according to any one of claims 1, 2 and 5, wherein it is a monoclonal antibody or antigen-binding fragment. 8. The antibody or antigen-binding fragment thereof according to claim 3, wherein it is a monoclonal antibody or antigen-binding fragment. 9. The antibody or antigen-binding fragment thereof according to claim 4, wherein it is a monoclonal antibody or antigen-binding fragment. 10. The antibody or antigen-binding fragment thereof according to claim 6, wherein it is a monoclonal antibody or antigen-binding fragment. 11. An antibody or antigen-binding fragment thereof according to any one of claims 1, 2, 5, 8, 9 and 10, wherein it is a human, humanized or chimeric antibody or antigen-binding fragment. 12. The antibody or antigen-binding fragment thereof according to claim 3, wherein it is a human, humanized or chimeric antibody or antigen-binding fragment. 13. The antibody or antigen-binding fragment thereof according to claim 4, wherein it is a human, humanized or chimeric antibody or antigen-binding fragment. 14. The antibody or antigen-binding fragment thereof according to claim 6, wherein it is a human, humanized or chimeric antibody or antigen-binding fragment. 15. The antibody or antigen-binding fragment thereof according to claim 7, wherein it is a human, humanized or chimeric antibody or antigen-binding fragment. 16. An antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 15. 17. An isolated nucleic acid encoding an antibody or antigen-binding fragment thereof according to any one of claims 1 to 15. 18. A vector comprising the nucleic acid according to claim 17. 19. Isolated cells expressing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 and/or containing a nucleic acid according to claim 17 or a vector according to claim 18. 20. The isolated cells of claim 19, wherein the cells are prokaryotic or eukaryotic cells. 21. A method for producing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 15, comprising culturing cells according to claim 20 and purifying said antibody or antigen-binding fragment. 22. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 15 or an antibody-drug conjugate according to claim 16. 23. The pharmaceutical composition according to claim 22 and a combination of one or more therapeutically active compounds. 24. Use of the pharmaceutical composition of claim 22 or the combination of claim 23 in the preparation of a medicament for treating cancer.