RU2784586C2 - Human antibodies binding to ror2 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: completely human antibody or its fragment is proposed, which specifically bind to an extracellular domain of a receptor tyrosine kinase-like orphan receptor 2 (ROR2). Antibody-drug conjugates including antibodies according to the invention are also proposed. In addition, the use of the specified conjugates in a method for the treatment of a neoplastic disease, as well as in a method for determination of a neoplastic disease or an immune disease in a patient or the risk of its development is disclosed.

EFFECT: invention can be used in cancer therapy.

17 cl, 11 dwg, 14 tbl, 10 ex

Description

The field of technology to which the present invention relates

The present invention relates to human antibodies that bind to ROR2, preferably human ROR2 (hROR2), and their fragments and conjugates, as well as their uses.

Background of the Invention

Cancer is one of the leading causes of death. It is a class of diseases caused by malignant transformation of healthy cells resulting from genetic changes like chromosomal translocations, mutations in tumor suppressor genes, exposure to transcription factors or growth factor receptors resulting in cell immortalization. If immortalization is combined with excessive proliferation, immortalized cells form tumors with or without metastasis (in the case of solid tumors) or leukemias and lymphomas (malignant neoplasms from blood cells). Defective apoptosis, or programmed cell death, may further contribute to the malignant transformation of cells leading to cancer.

The family of membrane-associated receptor tyrosine kinases, consisting of receptor tyrosine kinase-like orphan receptors 1 and 2 (ROR1 and ROR2), has been described to be specifically associated with specific cancers (Rebagay et al. (2012) Front Oncol. 2(34): 1-8 ; doi 10.3389/onc.2012.00034), while their expression is predominantly absent in healthy tissue with rare exceptions, for example, in the case of ROR1 (Balakrishnan et al. (2016) Clin Cancer Res. doi: 10.1158/1078-0432). It remains unclear whether ROR expression is functionally associated with tumor formation. However, due to the highly selective expression of members of the ROR family in tumors, they are important targets for anticancer therapeutics. Importantly, human ROR2 (hROR2) expression has been described on tumor cells in neuroblastoma, sarcoma (and especially osteosarcoma), renal cell carcinoma, breast cancer, testicular cancer, ovarian cancer, pancreatic cancer, kidney cancer, stomach cancer, prostate cancer, head and neck cancer, melanoma, squamous cell carcinoma, multiple myeloma and other cancers.

The receptor tyrosine kinase-like orphan receptors 1 and 2, ROR1 and ROR2, are the only two members that define a new family of receptor tyrosine kinases based on a common structural plan and some functional similarities. Both of the ROR1 and ROR2 proteins are type I singly spanning membrane transmembrane receptors with an extracellular domain (ECD) consisting of an immunoglobulin domain, a cysteine-rich frizzled domain, and a kringle domain. These three extracellular domains are followed by a transmembrane domain connecting the ECD to the intracellular portion of the protein containing the kinase domains (Rebagay et al. (2012) Frontiers Oncol. 2: 1-8).

The human ROR1 and ROR2 proteins are 58% homologous to each other, but each of the ROR proteins is highly conserved between species. This presents a challenge for the development of human ROR2-specific monoclonal antibodies, and very few antibodies are known.

Under normal physiological conditions, hROR2 is responsible for aspects of bone and cartilage growth during embryonic development. After birth, hROR2 expression is reduced and hROR2 is normally undetectable or expressed at very low levels in adult tissues. Only weak expression of hROR2 has been reported in stomach and thyroid tissue (Morioka et al. (2009) Cancer Sci. 100: 1227-1233). hROR2 has previously been considered as a target for the development of hROR2-specific antibodies (International Application WO 2013/103637 A1, International Application WO 2016/142768 A1).

There is a need for high quality anti-ROR2 antibodies that can be used as a basis for the development of targeted antibody therapeutics for ROR2-expressing cancers. In particular, there is also a need for anti-ROR2 antibodies having excellent design ease, including low aggregation propensity, and showing high thermostability, which are typically obtained using B cells from immunized animals. In addition, there is a need for antibodies that, in addition to binding to human ROR2, also bind to ROR2 in standard animal species used for toxicological studies such as mice, rats, rabbits and/or cynomolgus monkeys, and in particular bind to ROR2 of both the cynomolgus monkey and mouse ROR2, enabling the toxicological characterization of targeted antibody therapeutics in anticipation of human clinical trials. In addition, in the case of human therapy, there is a need for antibodies that are essentially fully human antibodies with the least risk of immunogenicity when administered systemically to human subjects. There is also a need for additional diagnostic tools to detect ROR2 expression in ROR2-associated disease states. The present invention addresses these and other needs.

Brief summary of the present invention

The present invention relates to fully human antibodies that specifically bind to the extracellular domain of the receptor tyrosine kinase-like orphan receptor 2 (ROR2). The present invention and the general advantages of its features will be discussed in detail below.

Brief description of the drawings

Fig. 1. Schematic representation of the process used to develop new human antibodies that target hROR2. (A) In the first step, human antibody transgenic mice (H2L2 mice) (described in WO 2010/070263 A1) were immunized exclusively with the extracellular domain bearing the Twin-Strep tag (hROR2-TwinStrep) at the C-terminus. (B) The number of hROR2-TwinStrep-binding spleen B cells was then increased by enriching cells from H2L2 immunized mice using magnetically activated cell sorting (MACS). (C) Libraries of transposable expression vectors for human IgG1 were generated from isolated spleen B cells by PCR cloning of the heavy and light chain variable domains from isolated hROR2-binding spleen B cells. (D) This was followed by transposition-mediated generation of cell libraries providing a display of full-length human IgG1 libraries stably expressing the PCR-cloned antibody libraries in immortalized pre-B cells. (E) FACS screening of cell-surface IgG1 antibodies for binding to hROR2 and isolation of single cell clones by FACS. (F) Functional screening of IgG1 antibodies from selected clonal supernatants. (G) PCR amplification of antibodies from selected clones.

Fig. 2. Detection of antibody titers to hROR2-ECD after immunization of H2L2 mice, named 1357, 1368 and 1359, with ECD-hROR2-TwinStrep. (A) before immunization and on day 7 (B), on day 28 (C) and on day 49 day (D) after the initial immunization.

Fig. 3. Antigen-specific FACS sorting per mouse based on IgG/IgK and IgM/IgK libraries: (A) H2L2 mouse 1357, (B) H2L2 mouse 1363 and (C) H2L2 mouse 1359.

Fig. 4. Determination of binding affinities to hROR2-ECD for selected monoclonal antibodies cloned and expressed using the genetic recombination techniques indicated, measured by surface plasmon resonance (SPR).

Fig. 5. Determination of cross-species cross-reactivity of recombinant monoclonal antibodies to hROR2 with mouse ROR2 and ROR2 of the cynomolgus monkey, which is assessed using the ELISA method.

Fig. 6. (A) Chemical structure of pentaglycine-modified PNU derivative ("G5-PNU"), (B) triglycine-modified PNU derivative ("G3-PNU").

Fig. 7. FACS analysis of hROR2 expression in the EMT-6 breast cancer cell line subjected to genetic engineering techniques to stably overexpress hROR2.

Fig. 8. In vitro evaluation of the cytolytic activity of antibody-drug conjugates containing PNU toxin (G5-PNU) site-specifically conjugated to IgH and IgL chains of selected anti-hROR2 antibodies as indicated using EMT-6 cells exposed to genetic techniques. engineered to stably express hROR2, according to Example 8.

Fig. 9. In Vitro Evaluation of Cytolytic Activity of Antibody-Drug Conjugates Containing Anti-hROR2 Monoclonal Antibody Clone MK-3B12 Conjugated to One of the PNU Toxins G3-PNU or G5-PNU Using Genetically Engineered EMT-6 Cells in order to stably express hROR2, according to example 9. Isotype-matched control ADC containing a clone of CD30-specific antibody - brentuximab (clone Ac10) and site-specifically conjugated G5-PNU, and CD30-expressing Karpas-299 cells used for control experiments as indicated.

Fig. 10. In Vitro Evaluation of Cytolytic Activity of Antibody Drug Conjugates Containing PNU Toxin (G5-PNU) Site-Specifically Conjugated to IgH and IgL Chains of Selected Anti-hROR2 Antibodies As Specified Using EMT-6 Cells Exposed to Genetic Techniques engineered to stably express hROR2, according to example 10.

Fig. 11. Determination of cross-species cross-reactivity of recombinant anti-hROR2 monoclonal antibodies with mouse ROR2 and cynomolgus ROR2 as assessed by ELISA.

Detailed disclosure of the present invention

Although the present invention has been illustrated and described in detail in the description of the drawings and the foregoing description, such illustration and description are considered illustrative or exemplary and not restrictive; the present invention is not limited to the disclosed embodiments. Other variants of the disclosed embodiments may be understood by those skilled in the art and carried out by them in the practical implementation of the claimed invention as a result of studying the drawings, the disclosure and the attached claims. In the claims, the word "comprising" does not exclude other elements or steps, and the singular forms do not exclude the plural. The fact that certain measurands are listed in different dependent claims does not in itself indicate that a combination of these measurands cannot be used to advantage. Any reference positions in the claims should not be construed as limiting the scope.

All amino acid sequences disclosed herein are in N-terminal to C-terminal orientation; all nucleic acid sequences disclosed herein are in 5' to 3' orientation.

In one aspect, the present invention relates to novel high affinity binding domains of fully human monoclonal antibodies that specifically bind to the extracellular domain of the tyrosine kinase-like orphan receptor 2 (ROR2). According to one embodiment, the ROR2 is a human ROR2 (hROR2).

Such monoclonal antibodies were selected from a variety of antibody libraries generated by stimulating transgenic human light and heavy chain variable domain mice (international application WO 2010/070263 A1, H2L2 mice) with the recombinant extracellular domain of the hROR2 antigen, followed by the creation and screening of cell libraries representing IgG1 from hROR2 stimulated H2L2 mice using DNA transfection vectors for transfection into 63-12 mouse pre-B cells according to WO 2014/013026 A1. Mammalian cells presenting antibodies according to international application WO 2014/013026 A1 were screened using antigen-specific cell sorting (FACS), including single cell sorting. Ultimately, antibodies secreted into the supernatant by sorted single cell clones were subjected to antigen binding by ELISA and cancer cell cytolysis assays by secondary antibodies (i.e., in which a toxin-conjugated secondary antibody binds to an antigen bound to the antibody to hROR2 on the cell surface, and is internalized to effect cytolysis). The coding sequences for the heavy and light chain variable domains were identified from selected cell clones exhibiting hROR2-specific binding and based on functional characterization involving cytolysis of hROR2-expressing target cells with a toxin-conjugated Fab-based secondary reagent. Using this strategy, novel fully human anti-hROR2 monoclonal antibodies of high quality and suitable functional properties were identified.

According to a second aspect of the present invention, antibody-drug conjugates (ADCs) based on said anti-ROR2 antibodies, antibody-based proteins or antigen-binding fragments thereof, are provided with one or more toxins, and in particular with the potent anthracycline toxin. In particular, such ADCs are obtained by site-specific conjugation, provided by enzymatic conjugation using a sortase enzyme, as disclosed in international application WO 2014/140317 A1, which is incorporated herein by reference. One particular highly potent anthracycline toxin has been disclosed in WO 2016/102679 A1, which is incorporated herein by reference.

According to a third aspect of the present invention, antibody-effector molecule (AEC) conjugates are provided based on said anti-ROR2 antibodies, antibody-based binding proteins or antigen-binding fragments thereof, conjugated to one or more labels.

In addition, the present invention relates to chimeric antigen receptors (CARs) and T cells that have been genetically engineered to express these CARs, i. CAR T cells using said anti-ROR2 antibodies, derivatives, modified formats or fragments.

In addition, the present invention relates to bi - or multispecific antibodies containing the binding domains of the disclosed antibodies to ROR2, to bi - or multispecific antibody formats containing at least one binding domain specific for another target, for example, without limitation, in relation to targets that recruit and/or activate immune system cells like T cells or NK cells. Such other binding domains may be specific for CD3, CD16, CD32, CD56, CD64, or other markers specific for T and NK cells.

In another aspect, the present invention provides isolated or substantially purified polynucleotides encoding an immunoglobulin heavy chain or immunoglobulin light chain variable region in a fully human anti-ROR2 antibody, antibody-based binding protein, or antigen-binding fragment thereof, and expression vectors carrying such polynucleotides, as well as host cells transformed or transfected with these polynucleotides, and methods for producing anti-ROR2 antibodies, antibody-based binding proteins, and antigen-binding fragments.

In another aspect, the present invention relates to a fully human antibody to ROR2, an antibody-based binding protein or antigen-binding fragment thereof, or an ADC, a bi- or multispecific antibody, or a chimeric antigen receptor (CAR) as described herein. , for use in the treatment of a subject who is suffering from a neoplastic disease, is at risk of developing a neoplastic disease, and/or has been diagnosed with a neoplastic disease.

According to yet another aspect, the present invention relates to a method of treating a subject suffering from a neoplastic disease, at risk of developing a neoplastic disease, and/or diagnosed with a neoplastic disease, with a human hROR2 antibody, an antibody-based binding protein, or an antigen-binding fragment thereof. , or an ADC, or a bi- or multispecific antibody, or a cell genetically engineered to express CAR as described herein.

According to yet another aspect, the present invention relates to a method for detecting a neoplastic disease or an immunopathological disease or disorder for which an anti-ROR2 antibody, an antibody-based binding protein or antigen-binding fragment thereof, or AEC is suitable.

Definitions

Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present invention pertains. In addition, the following definitions are provided to assist the reader in the practice of the present invention.

The term "human antibody" refers to an antibody that binds an antibody-based protein, a modified antibody format that retains the ability to bind to a target, or an antibody derivative or fragment that retains the ability to bind to a target that contains sequences derived from human immunoglobulins, due to whereby substantially all of the heavy and light chain CDR1 and CDR2 regions are of human origin, and substantially all of the heavy and light chain FR regions 1, 2, 3, and 4 correspond to those in the human immunoglobulin sequence with limited or no somatic mutations that can be introduced into separate variable domain sequences CDR1 and CDR2 and FR1, 2, 3 and 4 of the heavy and light chain.

The terms “antibody”, “antibody-based binding protein”, “modified antibody format that retains the ability to bind to a target”, “an antibody derivative or fragment that retains the ability to bind to a target” refer to the polypeptide(s) chain(s) which(which) exhibit(s) strong monovalent, bivalent or polyvalent binding to a given antigen, epitope or epitopes. The antibody protein-based proteins and antigen-binding fragments used in the present invention can be produced using any suitable technology, for example, hybridoma technology, ribosome display, phage display, gene shuffling libraries, semi-synthetic or fully synthetic libraries, or combinations thereof. Antibody binding protein-based antibodies and antigennegative fragments according to the present invention include intact antibodies and antibody fragments or antigennegative fragments that contain antigennegative sites of intact antibodies and retain the ability to bind to a cognate antigen. Unless otherwise defined herein, all peptide sequences, including all antibody and antigen-binding fragment sequences, are presented in the order N->C.

An intact antibody typically contains at least two heavy (H) chains (approximately 50-70 kDa) and two light (L) chains (approximately 25 kDa) linked by disulfide bonds. Known immunoglobulin genes encoding antibody chains include genes for the kappa, lambda, alpha, gamma, delta, epsilon and mu chain constant regions, as well as genes for a myriad of immunoglobulin variable regions. Light chains are either kappa or lambda. Heavy chains are of the gamma, mu, alpha, delta, or epsilon type, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. Each antibody heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. In the case of IgG, the heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region. The light chain constant region consists of a single domain, CL. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. The constant regions of antibodies can mediate the binding of immunoglobulin to tissues or host factors, including various cells of the immune system and the first component (Clq) of the classical complement system. Monoclonal antibodies (mAbs) are made up of identical antibody molecules.

The VH and VL regions of an antibody can be further subdivided into hypervariable regions, also referred to as complementarity determining regions (CDRs), in between which are more conserved framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino ring to carboxy ring in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The positions of the CDR and FR segments and the numbering system were determined, for example, according to the IMGT system (Lefranc MP et al., 2015) or the Kabat numbering scheme.

Antibodies binding protein-based antibodies and antigennegative fragments according to the present invention also encompasses single-chain antibodies. The term "single chain antibody" refers to a polypeptide that contains a VH domain and a VL domain in a polypeptide bridge, usually linked via a spacer peptide, and which may contain additional domains or amino acid sequences at the amino and/or carboxy ends. For example, a single chain antibody may contain a restriction segment for binding to a coding polynucleotide. By way of example, a single chain variable region fragment (scFv) is a single chain antibody. Compared to the VL and VH domains of the Fv fragment, which are encoded by separate genes, scFv has two domains joined (eg, by genetic recombination techniques) by a synthetic linker. This allows them to be obtained as a single protein chain, in which the VL and VH regions form a pair to form monovalent molecules.

Examples of antibody-based binding proteins are polypeptides in which antibody binding domains are combined with other polypeptides or polypeptide domains, e.g., alternative molecular backbones, Fc regions, other functional or binding domains of other polypeptides or antibodies, resulting in molecules with additional properties. binding, for example, bi - or multispecific proteins or antibodies. Such polypeptides can create a pattern of binding or functional domains that is not normally found in naturally occurring antibodies or antibody fragments.

Examples of antigen-binding fragments include (i) Fab fragment, a monovalent fragment consisting of VL, VH, CL and CHI domains; (ii) an F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of one arm of an intact antibody; (v) disulfide-stabilized Fvs (dsFv) that have an interchain disulfide bond genetically engineered between structurally conserved framework regions; (vi) a single domain antibody (dAb) that consists of a VH or VL domain (see, for example, Ward et al., Nature 341:544-546, 1989); and (vii) an isolated complementarity determining region (CDR) as a linear or cyclic peptide.

Science 240:1038-41, 1988. Стабилизированные дисульфидными связями Fv фрагменты (dsFv) можно получать с применением способов, описанных, например, в Reiter et al., Int. J. Cancer 67:113-23, 1996. Аналогично, однодоменные антитела (dAb) можно получать с помощью ряда способов, описанных, например, в Ward et al., Nature 341:544-546, 1989; и Cai and Garen, Proc. Natl. Acad. Sci. USA 93:6280-85, 1996. Однодоменные антитела верблюдовых можно получать с применением способов, хорошо известных в уровне техники, например, Dumoulin et al., Nat. Struct. Biol. 11:500-515, 2002; Ghahroudi et al., FEBS Letters 414:521-526, 1997; и Bond et al., J. Mol. Biol. 332:643-55, 2003. Другие типы антигенсвязывающих фрагментов (например, Fab, F(ab')₂ или Fd фрагменты) также можно легко получать с помощью обычно используемых в практике иммунологических методов. См., например, Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1998.Anti-ROR2 antibodies, antibody-based protein binding and antigen-binding fragments described herein can be generated by enzymatic or chemical modification of intact antibodies or synthesized de novo using recombinant DNA techniques. Methods for making these antibodies, antibody-based binding proteins, and antigen-binding molecules are well known in the art. In particular, scFv antibodies can be generated using the methods described in, for example, Bird et al., Science 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. sci. USA 85:5879-5883, 1988. Antibody Fv fragments can be generated as described in Skerra and

Science 240:1038-41, 1988. Disulfide-stabilized Fv fragments (dsFv) can be generated using the methods described, for example, in Reiter et al., Int. J. Cancer 67:113-23, 1996. Similarly, single-domain antibodies (dAbs) can be made using a number of methods, such as those described in, for example, Ward et al., Nature 341:544-546, 1989; and Cai and Garen, Proc. Natl. Acad. sci. USA 93:6280-85, 1996. Single domain camelid antibodies can be prepared using methods well known in the art, eg Dumoulin et al., Nat. Struct. Biol. 11:500-515, 2002; Ghahroudi et al., FEBS Letters 414:521-526, 1997; and Bond et al., J. Mol. Biol. 332:643-55, 2003. Other types of antigen-binding fragments (eg, Fab, F(ab') ₂ or Fd fragments) can also be readily prepared using commonly used immunological techniques. See, for example, Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1998.

Anti-ROR2 antibodies, antibody-based binding proteins or antigen-binding fragments of the present invention can be generated by any suitable technique, for example, using any suitable eukaryotic or non-eukaryotic expression system. In certain embodiments, an antibody, antibody-based binding protein, or antigen-binding fragment is produced using a mammalian-based expression system. Some specific techniques for obtaining antibodies, binding proteins based on antibodies or antigen-binding fragments or antigen-binding fragments according to the present invention are exemplified in this document. In some embodiments, antibodies, antibody-based protein binding or antigen-binding fragments of the present invention can be produced using a suitable non-eukaryotic expression system, such as a bacterial expression system. Bacterial expression systems can be used to generate fragments such as F(ab)2, Fv, scFv, IgGACH2, F(ab')2, scFv2CH3, Fab, VL, VH, scFv4, scFv3, scFv2, dsFv, Fv, scFv- Fc, (scFv)2, and diatel. Techniques for altering DNA coding sequences to obtain such fragments are known in the art.

The term "conservatively modified variant" refers to both amino acid sequences and nucleic acid sequences. With regard to specific nucleic acid sequences, the term "conservatively modified variants" refers to those nucleic acids that encode identical or substantially identical amino acid sequences, or in cases where the nucleic acid does not encode an amino acid sequence, substantially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids code for any given protein. For example, all of the codons GCA, GCC, GCG, and GCU code for the amino acid alanine. Thus, at each position at which an alanine is defined by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations", which are one kind of conservatively modified variations. Each nucleic acid sequence herein that encodes a polypeptide also describes each silent variation of the nucleic acid. The skilled artisan will appreciate that every codon in a nucleic acid (with the exception of AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, in each sequence described, each silent variation of a nucleic acid that encodes a polypeptide is contemplated.

In the case of polypeptide sequences, "conservatively modified variants" refers to a variant that has conservative amino acid substitutions, wherein amino acid residues are replaced with another amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the prior art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine , phenylalanine, tryptophan, histidine).

The terms "identical" or percent "identity" in the context of two or more nucleic acids or polypeptide sequences refers to two or more sequences or subsequences that are the same. Two sequences are "substantially identical" if two sequences share a certain percentage of amino acid residues or nucleotides that are the same (i.e. 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90% , 95%, or 99% identity at a specific site, or when undefined, over the entire sequence) when compared and aligned for best match in the comparison window or designated site when measured using one of the following algorithms for sequence comparison or manual alignment and visual inspection. Optionally, the identity exists in a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably in a region that is 100-500 or 1000 or more nucleotides in length (or 20, 50, 200 or more amino acids).

Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, using the local homology search algorithm from Smith and Waterman, Adv. Appl. Math. 2:482c, 1970; using the homologous region alignment algorithm from Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; using the similarity search method from Pearson and Lipman, Proc. Nat'l. Acad. sci. USA 85:2444, 1988; using implementations of these algorithms in computer programs (GAP, BESTFIT, FASTA, and TFASTA as part of the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wisconsin, USA); or by manual alignment and visual review (see, for example, Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (Ringbou ed., 2003)). Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively.

Percent (%) identity of peptide sequences can be calculated, for example, as 100 × [(identical positions)/min(TGA, TGB)], where TGA and TGB are the sum of the number of residues and positions of internal gaps in peptide sequences A and B when aligned , which minimizes TGA and TGB. See, for example, Russell et al, J. Mol. Biol, 244: 332-350 (1994).

Artificial T-cell receptors (also known as chimeric T-cell receptors, chimeric immunoreceptors, chimeric antigen receptors (CARs), or T-cell bodies (T-bodies)) are engineered receptors that confer arbitrary specificity on an effector immune cell. Typically, these receptors are used to confer specificity of a monoclonal antibody on a T cell; wherein the transfer of their coding sequence is facilitated by retroviral or lentiviral vectors or transposons. T cells engineered to express CAR are genetically engineered T cells equipped with chimeric receptors whose extracellular recognition portion consists of an antibody-derived recognition domain and whose intracellular region is derived from a lymphocyte-stimulating fragment (fragments). The structure of the prototype CARs is modular, designed to accommodate different functional domains and thereby enable selective specificity and controlled T cell activation. A preferred antibody-derived recognition portion is a single chain variable fragment (scFv) that combines specificity and binding residues in both the heavy and light chain variable regions of a monoclonal antibody, as well as other antibody-derived formats like Fab fragments, V domains etc., can be used to confer desired CAR specificity on T cells that have been genetically engineered to express CARs. The most common lymphocyte-activating fragments include a T-cell costimulatory domain (eg, CD28) in tandem with a T-cell-triggering (eg, CD3zeta) fragment, and other signaling domains like 4-1 BB can be used in the intracellular part CAR. By equipping effector lymphocytes (such as T cells and natural killer cells) with these chimeric receptors, the genetically engineered cell is retargeted with predetermined specificity to any desired target antigen without being restricted by HLA. CAR constructs are introduced ex vivo into T cells from peripheral lymphocytes of a given patient using retroviral or lentiviral vectors or transposons. After infusion of the resulting T cells, genetically engineered to express CARs, they migrate back to the patient, reach their target site, and when interacting with their target cell or tissue, they are activated and perform their predetermined effector function. Therapeutic targets for the CAR approach include cancer and HIV-infected cells or autoimmune effector cells.

The terms "treat", "treatment", "treatment", and "therapeutically effective" as used herein do not necessarily imply 100% or complete cure. More specifically, there are variable levels of treatment known to those skilled in the art to have a potential beneficial effect or therapeutic effect. In this regard, the method according to the present invention can provide any amount of any level of treatment. Moreover, the treatment provided by the method of the present invention may include the treatment of one or more conditions or symptoms of the disease being treated.

A "vector" is a replicon, such as a plasmid, phage, or cosmid, to which another polynucleotide segment can be attached in order to replicate the attached segment. Vectors capable of directing the expression of genes encoding one or more polypeptides are referred to as "expression vectors".

Detailed Disclosure of Embodiments

Aspects of the present invention relating to binding to human ROR2

In one aspect, the present invention relates to human anti-ROR2 antibodies, antibody-based binding proteins (including bi- or multispecific antibodies), antigen-binding fragments thereof, AECs, ADCs or CARs having the same binding specificity for ROR2 (and, in particular, in relation to hROR2 containing or consisting of SEQ ID NO. 1), i.e. binding to the same ROR2 epitope and/or competing for ROR2 binding with ROR2-specific antibodies comprising a pair of an immunoglobulin heavy chain variable region sequence and an immunoglobulin light chain variable region sequence according to Table 2. In particular, the present invention relates to human antibodies to ROR2, antibody-based binding proteins (including bi- or multispecific antibodies), their antigen-binding fragments, AECs, ADCs or CARs having the same binding specificity for ROR2 (and in particular for hROR2 containing or consisting of SEQ ID NO.1).

In one aspect, a fully human antibody or derivative, modified format, or fragment thereof is provided that specifically binds to the extracellular domain of an orphan receptor tyrosine kinase-like receptor 2 (ROR2).

In one embodiment, the antibody, derivative, modified format, or fragment is an antibody that binds an antibody-based protein, a modified antibody format that retains the ability to bind to a target, a derivative or fragment of an antibody that retains the ability to bind to a target.

In one embodiment, the antibody, derivative, modified format, or fragment (i) binds to the same ROR2 epitope and/or (ii) competes for binding to ROR2 with an antibody comprising an Ig heavy chain variable region sequence and a light chain variable region sequence Ig, respectively, presented in:

(i) SEQ ID NO:2 and SEQ ID NO:3;

(ii) SEQ ID NO: 4 and SEQ ID NO: 5;

(iii) SEQ ID NO:6 and SEQ ID NO:7;

(iv) SEQ ID NO:8 and SEQ ID NO:9;

(v) SEQ ID NO: 10 and SEQ ID NO: 11;

(vi) SEQ ID NO: 12 and SEQ ID NO: 13;

(vii) SEQ ID NO: 14 and SEQ ID NO: 15;

(viii) SEQ ID NO:16 and SEQ ID NO:17;

(ix) SEQ ID NO: 18 and SEQ ID NO: 19;

(x) SEQ ID N0:20 and SEQ ID NO:21;

(xi) SEQ ID NO:22 and SEQ ID NO:23 or

(xii) SEQ ID NO:24 and SEQ ID NO:25.

In accordance with one other embodiment, the antibody, derivative, modified format, or fragment comprises a heavy chain variable region sequence and a light chain variable region sequence, respectively, one or both of which are at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99% and most preferably 100% identical to the heavy chain variable region/light chain variable region sequence pairs above.

Binding competition assays include, but are not limited to, radioactive labeled immunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), sandwich ELISA assays, fluorescence-activated cell sorting (FACS) assays, and Biacore (SPR). When performing a competitive antibody assay between a control antibody and an antibody of interest, the reference sample can first be labeled with a detectable label, such as a fluorophore, biotin, or an enzymatic (or even radioactive) label, to allow subsequent identification. If the test antibody competes with a labeled control antibody, the intensity will decrease relative to the control reaction, which is carried out without the test antibody.

Methods for determining a binding epitope include, but are not limited to, assessing binding to an array of oligopeptides having (overlapping) amino acid sequences from the ROR2 sequence, deciphering the crystal or NMR structure of an antibody, antibody-based binding protein, or antigen-binding fragment thereof to ROR2 by assessing loss binding antibodies that bind antibody-based proteins, their antigen-binding fragments to ROR2 containing one or more amino acid mutations ("high-throughput mutagenesis"), and using hydrogen-deuterium exchange to assess the solvent-accessible surface of the ROR2-antibody complex, an antibody-based binding protein or antigen-binding fragment (Abbott M. et al, 2014).

According to a preferred embodiment, fully human anti-ROR2 antibodies binding antibody-based proteins, antigen-binding fragments thereof, AECs, ADCs or CARs, having the same binding specificity for ROR2 (and in particular for hROR2 containing or consisting of SEQ ID NO. 1), i.e. binding to the same ROR2 epitope and/or competing for binding to ROR2 with ROR2-specific antibodies containing a pair of an immunoglobulin heavy chain variable region (HCVR Ig) sequence and an immunoglobulin light chain variable region (LCVR Ig) sequence, respectively, as shown in : (i) SEQ ID NO:2 and SEQ ID NO:3; (ii) SEQ ID NO:6 and SEQ ID NO:7; (iii) SEQ ID NO:10 and SEQ ID NO:ll; (iv) SEQ ID NO:12 and SEQ ID NO:13; (v) SEQ ID NO:14 and SEQ ID NO:15; (vi) SEQ ID NO:16 and SEQ ID NO:17; (vii) SEQ ID NO:4 and SEQ ID NO:5; (viii) SEQ ID NO:8 and SEQ ID NO:9; (ix) SEQ ID NO:18 and SEQ ID NO:19; (x) SEQ ID NO:24 and SEQ ID NO:25; (xi) SEQ ID NO:20 and SEQ ID NO:21; and (xii) SEQ ID NO:22 and SEQ ID NO:23, and more preferably as shown in (i) SEQ ID NO:2 and SEQ ID NO:3; (ii) SEQ ID NO:8 and SEQ ID NO:9; and (iii) SEQ ID N0:20 and SEQ IDNO:21.

In various embodiments, an antibody, an antibody-based protein, an antigen-binding fragment thereof, an AEC, an ADC, or a CAR is considered to be competitive with a control antibody if it reduces binding of the control antibody by at least about 20% or more, e.g., at least approximately 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or greater, or a percentage between any of the above, at a control antibody concentration , which is 80% of the maximum binding under the conditions of the specific assay used, and the concentration of the test antibody or antigen-binding fragment, which is 10-fold greater than the concentration of the control antibody.

Preferred Embodiments Related to the Foregoing Aspects of the Present Invention

In accordance with any of the above aspects of the present invention, a fully human anti-ROR2 antibody, antibody-based binding protein, antigen-binding fragment thereof, AEC, ADC or CAR preferably binds to a ROR2 containing or consisting of SEQ ID NO. 1 with a K _d of less than 100 nM, more preferably less than 75 nM, even more preferably less than 50 nM, and most preferably less than 25 nM.

In accordance with any of the above aspects of the present invention, a fully human anti-ROR2 antibody, antibody-based binding protein, antigen-binding fragment thereof, AEC, ADC or CAR preferably binds to a ROR2 containing or consisting of SEQ ID NO. 1 with k _on greater than 1×10 ⁴ M ^-1 s ^-1 , more preferably greater than 1×10 ⁵ M ^-1 s ^-1 , even more preferably greater than l×10 ⁶ M ^-1 s ^-1 .

In accordance with any of the above aspects of the present invention, a fully human anti-ROR2 antibody, antibody-based binding protein, antigen-binding fragment thereof, AEC, ADC or CAR preferably binds to a ROR2 containing or consisting of SEQ ID NO. 1 with k _0ff less than 1x10 ^-2 s ^-1 , more preferably less than 1x10 ^-3 s ^-1 and even more preferably less than 1x10 ^-4 s ^-1 .

Binding affinity is usually expressed in terms of the association or dissociation equilibrium constants (K _a or K _d , respectively), which in turn are the reciprocals of the dissociation and association rate constants (k _0ff and k _on , respectively). Thus, equivalent affinities can correspond to different rate constants, as long as the ratio of rate constants remains the same. Binding affinities and/or rate constants can be determined using techniques well known in the art or described herein, such as, for example, ELISA, isothermal titration calorimetry (ITC), Biacore (SPR), biolayer interferometry, or fluorescence polarization.

The present invention also relates to fully human anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADC, AEC or CAR, containing a V _H chain containing three CDRs, wherein:

CDR No. 1 V _H contains or consists of the peptide sequence GYSISSGYY (SEQ ID NO. 26) or GX ₁ X ₂ FX ₃ X ₄ X ₅ X ₆ (SEQ ID NO. 67), in which X ₁ =F or G, X ₂ =T or S, X ₃ =R or S, X ₄ =S, T, R or G, X ₅ =H, Y or Q, X ₆ =G, Y or R; and/or

CDR #2 V _H contains or consists of the peptide sequence IYQSGST (SEQ ID NO. 27), INHSRTT (SEQ ID NO. 59), INHSGIT (SEQ ID NO. 93) or IX ₇ X ₈ DGX ₉ X ₁₀ K (SEQ ID NO. 68) wherein X ₇ ═W or K, X ₈ ═Y, N, F or Q, X ₉ ═S or T, X ₁₀ ═K, N or E; and/or

V _H CDR #3 contains or consists of a peptide sequence selected from: CAREDRAGWYPFDCW (SEQ ID NO. 28), CARVGAGLYLDYW (SEQ ID NO. 33), CAREGSGWYDYYYGMDVW (SEQ ID NO. 40), CQHYNTYSRTF (SEQ ID NO. 62) , CARPGIAMTGLDYW (SEQ ID NO. 37), CARGGDQWLVPFDNW (SEQ ID NO. 95), CARVAAALHFHYW (SEQ ID NO. 47), CARGGEQWLVPFDYW (SEQ ID NO. 60), CARPGVAMTGLDLW (SEQ ID NO. 43), CIRVKFGDLYFQHW (SEQ ID NO. 49), CVRVRFGELYFQHW (SEQ ID NO. 44), CARDGYRNGWHIPEDYW (SEQ ID NO. 65), CARMGAINRGGGGFDYW (SEQ ID NO. 52), and CARDKGEWFGELRYYYYGMDVW (SEQ ID NO. 55).

The present invention also relates to fully human anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADC, AEC or CAR, containing a V _H chain containing three CDRs, wherein:

CDR No. 1 V _H contains or consists of the peptide sequence GYSISSGYY (SEQ ID NO. 26) or GX ₁ X ₂ FX ₃ X ₄ X ₅ X ₆ (SEQ ID NO. 67), in which X ₁ =F or G, X ₂ =T or S, X ₃ =R or S, X ₄ =S, T, R or G, X ₅ =H, Y or Q, X ₆ =G, Y or R; and

CDR No. 2 V _H contains or consists of the peptide sequence IYQSGST (SEQ ID NO. 27), INHSRTT (SEQ ID NO. 59) or IX7X8DGX9X10K (SEQ ID NO. 68), in which X ₇ =W or K, X ₈ = Y, N, F or Q, X ₉ =S or T, X ₁₀ =K, N or E; and

V _H CDR #3 contains or consists of a peptide sequence selected from: CAREDRAGWYPFDCW (SEQ ID NO. 28), CARVGAGLYLDYW (SEQ ID NO. 33), CAREGSGWYDYYYGMDVW (SEQ ID NO. 40), CQHYNTYSRTF (SEQ ID NO. 62) , CARPGIAMTGLDYW (SEQ ID NO. 37), CARGGDQWLVPFDNW (SEQ ID NO. 95), CARVAAALHFHYW (SEQ ID NO. 47), CARGGEQWLVPFDYW (SEQ ID NO. 60), CARPGVAMTGLDLW (SEQ ID NO. 43), CIRVKFGDLYFQHW (SEQ ID NO. 49), CVRVRFGELYFQHW (SEQ ID NO. 44), CARDGYRNGWHIPEDYW (SEQ ID NO. 65), CARMGAINRGGGGFDYW (SEQ ID NO. 52), and CARDKGEWFGELRYYYYGMDVW (SEQ ID NO. 55).

The present invention also relates to fully human anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADC, AEC or CAR, containing a V _H chain containing three CDRs, wherein:

CDR #1 V _H contains or consists of the peptide sequence GYSISSGYY (SEQ ID NO. 26), GFTFRSHG (SEQ ID NO. 31), GGSFSGYY (SEQ ID NO. 58), GFTFSSQR (SEQ ID NO. 63), or GFTFX3X4YG (SEQ ID NO. 71), in which X ₃ =R or S, and X ₄ =S, T or R, and preferably contains or consists of the peptide sequence GYSISSGYY (SEQ ID NO. 26), GFTFRSYG (SEQ ID NO 36 ), GFTFRTYG (SEQ ID NO 94), GFTFSRYG (SEQ ID NO 45), GFTFRSHG (SEQ ID NO. 31), GFTFSSYG (SEQ ID NO. 38), GGSFSGYY (SEQ ID NO. 58), or GFTFSSQR (SEQ ID NO. 58) 63), and more preferably contains or consists of the peptide sequence GYSISSGYY (SEQ ID NO. 26) or GFTFSSYG (SEQ ID NO. 38); and

CDR #2 V _H contains or consists of the peptide sequence IYQSGST (SEQ ID NO. 27), IWYDGSKK (SEQ ID NO 32), IWYDGSNK (SEQ ID NO 46), IWNDGSNK (SEQ ID NO. 39), IWFDGTNK (SEQ ID NO. 54), INHSRTT (SEQ ID NO. 59) or IKQDGSEK (SEQ ID NO. 64) and preferably contains or consists of the peptide sequence IYQSGST (SEQ ID NO. 27), IWNDGSNK (SEQ ID NO. 39) or IWFDGTNK (SEQ ID NO. 54); and

V _H CDR #3 contains or consists of a peptide sequence selected from: CAREDRAGWYPFDCW (SEQ ID NO. 28), CARPGIAMTGLDYW, CARPGVAMTGLDLW (DEQ ID NO. 43), CVRVRFGELYFQHW (SEQ ID NO. 44), CARVAAALHFHYW (SEQ ID NO. 47), CIRVKFGDLYFQHW (SEQ ID NO. 49), CARVGAGLYLDYW (SEQ ID NO. 33), CARPGIAMTGLDYW (SEQ ID NO. 37), CAREGSGWYDYYYGMDVW (SEQ ID NO. 40), CARDKGEWFGELRYYYYGMDVW (SEQ ID NO. 55), CARGGEQWLVPFDYW ( SEQ ID NO. 60) or CARDGYRNGWHIPEDYW (SEQ ID NO. 65) and preferably contains or consists of a peptide sequence selected from: CAREDRAGWYPFDCW (SEQ ID NO. 28), CAREGSGWYDYYYGMDVW (SEQ ID NO. 40) or CARDKGEWFGELRYYYYGMDVW (SEQ ID NO.55).

In accordance with any of the above aspects of the present invention, anti-ROR2 antibodies, antibody-based protein-binding proteins, antigen-binding fragments thereof, ADC, AEC or CAR, containing a V _H chain containing three CDRs, wherein:

CDR #1 V _H contains or consists of a peptide sequence selected from the HC CDR1 sequences listed in Table 3; and/or

CDR #2 V _H contains or consists of a peptide sequence selected from the HC CDR2 sequences listed in Table 3; and/or

CDR #3 V _H contains or consists of a peptide sequence selected from the HC CDR3 sequences listed in Table 3.

In accordance with any of the above aspects of the present invention, anti-ROR2 antibodies, antibody-based protein-binding proteins, antigen-binding fragments thereof, ADC, AEC or CAR, containing a V _H chain containing three CDRs, wherein:

CDR #1 V _H contains or consists of a peptide sequence selected from the HC CDR1 sequences listed in Table 3; and

CDR #2 V _H contains or consists of a peptide sequence selected from the HC CDR2 sequences listed in Table 3; and

CDR #3 V _H contains or consists of a peptide sequence selected from the HC CDR3 sequences listed in Table 3.

In accordance with any of the above aspects of the present invention, antibodies to ROR2, antibody-based protein binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs containing a V _H chain, contain three CDRs, CDR #1 V _H , CDR #2 V _H and CDR #3 V _H contains or consists of a peptide sequence corresponding to a predetermined set of HC CDR1, CDR2 and CDR3 shown in Table 3.

In accordance with any of the above aspects of the present invention, antibodies to ROR2, antibody-based protein binding proteins, antigen-binding fragments thereof, ADCs, AECs or CARs containing a V _H chain, contain three CDRs, CDR #1 V _H , CDR #2 V _H and CDR #3 V _H contain or consist of a peptide sequence corresponding to a given set of CDR1, CDR2 and CDR3 HC from MK-3 B12, GK-5A1, GK-21D3, MK-24C10, MK-24F9 or GK-22G12 and, more preferably from MK-3 B12, GK-5A1 or MK-24C10 shown in Table 3.

The present invention also relates to antibodies to ROR2, binding proteins based on antibodies, their antigen-binding fragments, ADC, AEC or CAR, containing a V _L chain containing three CDRs, wherein:

CDR No. 1 V _L contains or consists of the peptide sequence QSLLHSNGYNY (SEQ ID NO. 56), QSIDNW (SEQ ID NO. 48) or QX ₁₁ ISX ₁₂ X ₁₃ (SEQ ID NO. 69), in which X ₁₁ =S, T or G, X ₁₂ =S, N or H and X ₁₃ =W or Y; and/or

CDR #2 V _L contains or consists of a peptide sequence of KAS, AAS or LGS; and/or

CDR No. 3 V _L contains or consists of the peptide sequence CX ₁₄ X ₁₅ X ₁₆ X ₁₇ X ₁₈ X ₁₉ X ₂₀ X ₂₁ X ₂₂ F (SEQ ID NO. 70), in which X ₁₄ =Q or M, X ₁₅ = K, H or Q, X ₁₆ =H, Y or A, X ₁₇ =N or L, X ₁₈ =R, T, Q, S or N; X ₁₉ =A, Y or T; X ₂₀ \u003d P, S or W, X ₂₁ \u003d W, R, Y or absent, X ₂₂ \u003d S or T.

The present invention also relates to antibodies to ROR2, binding proteins based on antibodies, their antigen-binding fragments, ADC, AEC or CAR containing a V _L chain containing three CDRs, wherein:

CDR No. 1 V _L contains or consists of the peptide sequence QSLLHSNGYNY (SEQ ID NO. 56), QSIDNW (SEQ ID NO. 48) or QX ₁₁ ISX ₁₂ X ₁₃ (SEQ ID NO. 69), in which X ₁₁ =S, T or G, X ₁₂ =S, N or H and X ₁₃ =W or Y; and

CDR #2 V _L contains or consists of a peptide sequence of KAS, AAS or LGS; and

CDR No. 3 V _L contains or consists of the peptide sequence CX ₁₄ X ₁₅ X ₁₆ X ₁₇ X ₁₈ X ₁₉ X ₂₀ X ₂₁ X ₂₂ F (SEQ ID NO. 70), in which X ₁₄ =Q or M, X ₁₅ = K, H or Q, X ₁₆ =H, Y or A, X ₁₇ =N or L, X ₁₈ =R, T, Q, S or N; X ₁₉ =A, Y or T; X ₂₀ =P, S or W, X ₂₁ =W, R, Y or absent, X ₂₂ =S or T.

The present invention also relates to antibodies to ROR2, binding proteins based on antibodies, their antigen-binding fragments, ADC, AEC or CAR containing a V _L chain containing three CDRs, wherein:

CDR #1 V _L contains or consists of the peptide sequence QSISSW (SEQ ID NO. 29), QTISNW (SEQ ID NO. 34), QSIDNW (SEQ ID NO. 48), QGISNY (SEQ ID NO. 50), QSLLHSNGYNY (SEQ ID NO. 56) or QSISHW (SEQ ID NO. 61) and preferably contains or consists of the peptide sequence QSISSW (SEQ ID NO. 29) or QSLLHSNGYNY (SEQ ID NO. 56); and

CDR #2 V _L contains or consists of a KAS, LGS or AAS peptide sequence and preferably contains or consists of a KAS or LGS peptide sequence; and

CDR #3 V _L contains or consists of the peptide sequence CQQYNNYWTF (SEQ ID NO. 30), CQQYNSYWTF (SEQ ID NO. 41), CQQYNSYSYSF (SEQ ID NO. 42), CQQYNSYSYSF (SEQ ID NO. 44), CQQYNSYSYTF (SEQ ID NO. 35), CQKYNSAPYTF (SEQ ID NO. 51), CARMGAINRGGGGFDYW (SEQ ID NO. 52), CQKYNSAPWTF (SEQ ID NO. 53), CMQALQTPYTF (SEQ ID NO. 57), CQHYNTYSRTF (SEQ ID NO. 62) or CQKHNRAPWTF (SEQ ID NO. 66) and preferably contains, or consists of, the peptide sequence CQQYNNYWTF (SEQ ID NO. 30), CQQYNSYWTF (SEQ ID NO. 41) or CMQALQTPYTF (SEQ ID NO. 57).

In accordance with any of the above aspects of the present invention, fully human anti-ROR2 antibodies that bind antibody-based proteins, antigen-binding fragments thereof, ADC, AEC or CAR, containing a V _L chain containing three CDRs, wherein:

CDR #1 V _L contains or consists of a peptide sequence selected from the CDR1 LC sequences listed in Table 3; and/or

CDR #2 V _L contains or consists of a peptide sequence selected from the CDR2 LC sequences listed in Table 3; and/or

CDR #3 V _L contains or consists of a peptide sequence selected from the CDR3 LC sequences listed in Table 3.

In accordance with any of the above aspects of the present invention, fully human anti-ROR2 antibodies that bind antibody-based proteins, antigen-binding fragments thereof, ADC, AEC or CAR, containing a V _L chain containing three CDRs, wherein:

CDR #1 V _L contains or consists of a peptide sequence selected from the CDR1 LC sequences listed in Table 3; and

CDR #2 V _L contains or consists of a peptide sequence selected from the CDR2 LC sequences listed in Table 3; and

CDR #3 V _L contains or consists of a peptide sequence selected from the CDR3 LC sequences listed in Table 3.

In accordance with any of the above aspects of the present invention, fully human antibodies to ROR2, binding proteins based on antibodies, their antigen-binding fragments, ADC, AEC or CAR containing the V _L chain, contain three CDRs, with CDR # 1 V _L , CDR # 2 V _L and CDR #3 V _L contain or consist of a peptide sequence corresponding to a predetermined set of CDR1, CDR2 and CDR3 LC presented in Table 3.

In accordance with any of the above aspects of the present invention, antibodies to ROR2, antibody-based protein-binding proteins, antigen-binding fragments thereof, ADC, AEC or CAR containing a V _L chain, contain three CDRs, with CDR No. 1 V _L , CDR No. 2 V _L and CDR #3 V _L contain or consist of a peptide sequence corresponding to the set of CDR1, CDR2 and CDR3 LC of MK-3 B12, GK-5A1, GK-21D3, MK-24C10, MK-24F9 or GK-22G12, and, more preferably from MK-3 B12, GK-5A1 or MK-24C10 shown in Table 3.

In accordance with any of the above aspects of the present invention, antibodies to ROR2 binding proteins based on antibodies, their antigen-binding fragments, ADC, AEC or CAR, contain a V _L and V _H chain, each of which contains three CDRs containing or consisting, according to with Table 3, from the peptide sequence of the corresponding CDRs from: MK-3 B12, MK-7C3, GK-1E5, GK-5E1, GK-2G8, GK-5A1, GK-6B10, GK-5G12, GK-21D3, MK- 24C10, MK-24F9 or GK-22G12 and preferably from: MK-3 B12, GK-5A1, GK-21D3, MK-24C10, MK-24F9 or GK-22G12 and more preferably from: Mk-3 B12, GK-5A1 or MK-24C10.

In accordance with any of the above aspects of the present invention, antibodies to ROR2 binding proteins based on antibodies, their antigen-binding fragments, ADC, AEC or CAR, contain a V _L and V _H chain, each of which contains three CDRs containing or consisting, according to with Table 3, from the peptide sequence of the corresponding CDRs from: MK-3 B12, GK-1E5, GK-2G8, GK-5E1, GK-6 B10 or GK-5G12, or from: GK-5A1, GK-21D3, MK- 24C10, MK-24F9, MK-7C3 or GK-22G12, or more preferably from: MK-3 B12, GK-5A1, GK-21D3, MK-24C10, MK-24F9 or GK-22G12, or more preferably from: MK-3 B12, GK-5A1 or MK-24C10.

In accordance with one embodiment, an antibody, an antibody-based protein binding, a modified antibody format that retains the ability to bind to a target, an antibody derivative or fragment that retains the ability to bind to a target, is cross-reactive to (i) human ROR2 (hROR2) and (ii) at least one of cynomolgus monkey ROR2 (cROR2) and mouse RoR2 (mROR2).

When used in the context of this document, the term "cross-reactivity" means that an antibody that binds an antibody-based protein, a modified antibody format that retains the ability to bind to a target, an antibody derivative or fragment that retains the ability to bind to a target, is capable of binding ( i) a human ROR2 (hROR2); and (ii) at least one of a cynomolgus monkey ROR2 (cROR2) and a mouse RoR2 (mROR2) with sufficient affinity.

This cross-reactivity has significant advantages in preclinical applications, since the same antibody that has been used in mouse and cynomolgus monkey studies can be further used in clinical trials. In addition, this cross-reactivity has significant advantages in diagnostics as well as in scientific applications.

In accordance with one embodiment, the antibody, the antibody-based binding protein, the modified antibody format that retains the ability to bind to a target, the antibody derivative or fragment that retains the ability to bind to a target, contains one of the following sets of CDRs:

a) heavy chain CDRs 1-3 as set forth in SEQ ID NOs 38, 39 and 40 and light chain CDRs 1 and 3 as set forth in SEQ ID NOs 29 and 41, with light chain CDR 2 having the sequence KAS;

b) heavy chain CDRs 1-3 as set forth in SEQ ID NOs 26-28 and light chain CDRs 1-3 as set forth in SEQ ID NOs 29, 30 and 73;

c) heavy chain CDRs 1-3 as set forth in SEQ ID NOs 94, 32 and 44 and light chain CDRs 1 and 3 as set forth in SEQ ID NOs 29 and 42, with light chain CDR 2 having the sequence KAS;

d) heavy chain CDRs 1-3 as set forth in SEQ ID NOs 38, 46 and 52, and light chain CDRs 1 and 3 as set forth in SEQ ID NOs 50 and 53, with light chain CDR 2 having the sequence AAS;

e) heavy chain CDRs 1-3 as set forth in SEQ ID NOs 38, 54 and 55 and light chain CDRs 1 and 3 as set forth in SEQ ID NOs 56 and 57, with light chain CDR 2 having the LGS sequence;

f) heavy chain CDRs 1-3 as set forth in SEQ ID NOs 58-60 and light chain CDRs 1 and 3 as set forth in SEQ ID NOs 61 and 62, with light chain CDR 2 having the sequence KAS;

g) heavy chain CDRs 1-3 as set forth in SEQ ID NOs 63-65 with light chain CDRs 1 and 3 as set forth in SEQ ID NOs 50 and 66 with light chain CDRs 2 having the sequence AAS.

The CDRs are contained in a suitable protein scaffold to be capable of binding to hROR2 as well as at least one of the cynomolgus monkey ROR2 (cROR2) and mouse RoR2 (mROR2) with sufficient affinity.

In one embodiment, the antibody, the antibody-based protein binding, the modified antibody format that retains the ability to bind to a target, the antibody derivative or fragment that retains the ability to bind to a target, comprises one of the following sequence pairs:

a) the sequence of the heavy chain variable region of the GK-5A1 antibody shown in SEQ ID NO. 8 and the GK-5A1 antibody light chain variable region sequence shown in SEQ ID NO. 9,

b) the sequence of the variable region of the heavy chain of the antibody MK-3 B12, presented in SEQ ID NO. 2 and the light chain variable region sequence of the MK-3B12 antibody shown in SEQ ID NO. 3,

c) the sequence of the heavy chain variable region of the GK-5E1 antibody shown in SEQ ID NO. 12 and the GK-5E1 antibody light chain variable region sequence shown in SEQ ID NO. 13,

d) the sequence of the heavy chain variable region of the GK-21D3 antibody as shown in SEQ ID NO. 18 and the GK-5E1 antibody light chain variable region sequence shown in SEQ ID NO. 19,

e) the sequence of the variable region of the heavy chain of the antibody MK-24C10, presented in SEQ ID NO. 20 and the light chain variable region sequence of the MK-24C10 antibody shown in SEQ ID NO. 21,

f) the sequence of the variable region of the heavy chain of the antibody MK-24F9, presented in SEQ ID NO. 22 and the light chain variable region sequence of the MK-24F9 antibody shown in SEQ ID NO. 23,

g) the sequence of the variable region of the heavy chain of the antibody GK-22G12, presented in SEQ ID NO. 24 and the GK-22G12 antibody light chain variable region sequence shown in SEQ ID NO. 25.

In one embodiment, the antibody, antibody-based protein-based, modified antibody format that retains the ability to bind to a target, an antibody derivative or fragment that retains the ability to bind to a target, bi- or multispecific molecules comprise:

• a first moiety that binds to the extracellular domain of the receptor tyrosine kinase-like orphan receptor 2 (ROR2), and

• at least a second portion that binds to an effector antigen selected from the group consisting of CD3, CD16, NKG2D, NKp46, CD2, CD28 and/or CD25.

Some sequences are shown below:

In accordance with all of the above aspects of the present invention, antibodies to ROR2, antibody-based protein binding proteins, antigen-binding fragments thereof, ADC, AEC or CAR preferably contain a pair of heavy chain/light chain variable regions, the sequences of which are substantially identical to the variable region of any pair from the table 2 and, in particular, sequences selected from the following pairs (table 2): (i) SEQ ID NO:2 and SEQ ID NO:3; (ii) SEQ ID NO:4 and SEQ ID NO:5; (iii) SEQ ID NO:6 and SEQ ID NO:7; (iv) SEQ ID NO:8 and SEQ ID NO:9; (v) SEQ ID N0:10 and SEQ ID NO:11; (vi) SEQ ID NO:12 and SEQ ID NO:13; (vii) SEQ ID NO:14 and SEQ ID NO:15; (vii) SEQ ID NO:16 and SEQ ID NO:17; (ix) SEQ ID NO:18 and SEQ ID NO:19; (x) SEQ ID N0:20 and SEQ ID NO:21; (xi) SEQ ID NO:22 and SEQ ID NO:23 or (xii) SEQ ID NO:24 and SEQ ID NO:25. According to a preferred embodiment, the fully human anti-ROR2 antibody or antigen binding fragment comprises a heavy chain/light chain variable region pair whose sequences are substantially identical to sequences selected from the following pairs (Table 2): (i) SEQ ID NO:2 and SEQ ID NO:3; (ii) SEQ ID NO:8 and SEQ ID NO:9; (iii) SEQ ID NO:18 and SEQ ID NO:19; (iv) SEQ ID NO:20 and SEQ ID NO:21; (v) SEQ ID NO:22 and SEQ ID NO:23 or (vi) SEQ ID NO:24 and SEQ ID NO:25. According to a preferred embodiment, the fully human anti-ROR2 antibody or antigen binding fragment comprises a heavy chain/light chain variable region pair whose sequences are substantially identical to those selected from the following pairs (Table 2): (i) SEQ ID NO:2; SEQ ID NO:3; (ii) SEQ ID NO:8 and SEQ ID NO:9; and (iii) SEQ ID NO:20 and SEQ ID NO:21.

According to a preferred embodiment, "substantially identical" means at least 90%, preferably 95%, more preferably 100% identical to the corresponding variable region amino acid sequence identified in the corresponding SEQ ID NO.

In accordance with some embodiments, the present invention provides fully human anti-ROR2 antibodies that bind antibody-based proteins, antigen-binding fragments thereof, which are conservatively modified variants of the human anti-ROR2 antibodies illustrated herein. Typically, the variable regions of these variants have an amino acid sequence that is identical to one of these illustrated sequences except for conservative substitutions at one or more amino acid residues.

In accordance with all of the above aspects of the present invention, human antibodies that bind antibody-based proteins, their antigen-binding fragments are preferably IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, IgM, F(ab)2, Fv, scFv , IgGACH2, F(ab')2, scFv2CH3, Fab, VL, VH, scFv4, scFv3, scFv2, dsFv, Fv, scFv-Fc, (scFv)2, non-impoverishing IgG, diabody or bivalent antibody. In accordance with a more preferred embodiment of human antibodies that bind antibody-based proteins, their antigen-binding fragments are IgG1, IgG2, IgG3, IgG4, IgM.

In the case where human antibodies binding antibody-based proteins or antigen-binding fragments thereof (or ADCs or CARs derived therefrom) are bivalent, it is preferred that they bind to both human ROR2 and human CD3.

According to a preferred embodiment, anti-ROR2 antibodies, antibody-based protein binding proteins, antigen-binding fragments thereof, ADC, AEC or CAR according to the present invention bind to human ROR2 and cynomolgus ROR2 and compete for binding with an antibody containing GK-5A1 variable domains. , MK-3 B12, GK-5E1, MK-21D3, MK-24C10, MK-24F9 or MK-22G12 and preferably with an antibody containing variable domains GK-5A1, MK-3 B12, MK-24C10, MK- 24F9 or MK-22G12.

According to a preferred embodiment of an anti-ROR2 antibody, the antibody-based binding protein, antigen-binding fragments thereof, ADC, AEC or CAR of the present invention bind to human ROR2 and cynomolgus ROR2. According to a preferred embodiment, anti-ROR2 antibodies, antibody-based protein-binding proteins, antigen-binding fragments thereof, ADC, AEC or CAR bind to cynomolgus monkey ROR2 and human ROR2 and contain CDRs from GK-5A1, MK-3B12, GK-5E1, MK-21D3, MK-24C10, MK-24F9 or MK-22G12 and preferably from GK-5A1, MK-3B12, MK-24C10, MK-24F9 or MK-22G12. According to a preferred embodiment, the anti-ROR2 antibodies, antibody-based protein-binding proteins, antigen-binding fragments thereof, ADC, AEC or CAR according to the present invention bind to cynomolgus monkey ROR2 and to human ROR2 and contain variable domains from GK-5A1, MK-3B12, GK-5E1, MK-21D3, MK-24C10, MK-24F9 or MK-22G12 and preferably from GK-5A1, MK-3 B12, MK-24C10, MK-24F9 or MK-22G12.

According to a preferred embodiment, anti-ROR2 antibodies, antibody-based protein binding proteins, antigen-binding fragments thereof, ADC, AEC or CAR according to the present invention bind to human ROR2 and mouse ROR2 and compete for binding with an antibody containing variable domains from GK-5A1 , MK-24C10 or MK-24F9, and preferably with an antibody containing variable domains from GK-5A1.

According to a preferred embodiment, the anti-ROR2 antibodies, antibody-based protein binding proteins, antigen-binding fragments thereof, ADC, AEC or CAR of the present invention bind to murine ROR2 and to human ROR2. According to a preferred embodiment, antibodies to ROR2, antibody-based binding proteins, antigen-binding fragments thereof, ADC, AEC or CAR according to the present invention bind to murine ROR2 and human ROR2 and contain a CDR from GK-5A1, MK-24C10 or MK- 24F9 and preferably from GK-5A1. According to a preferred embodiment, anti-ROR2 antibodies, antibody-based protein binding proteins, antigen-binding fragments thereof, ADC, AEC or CAR according to the present invention bind to murine ROR2 and human ROR2 and contain variable domains from GK-5A1, MK-24C10 or MK -24F9 and preferably from GK-5A1.

Cross-reactivity of the antibodies of the present invention with murine ROR2 is particularly surprising given that these antibodies are mouse-derived.

According to a preferred embodiment, anti-ROR2 antibodies, antibody-based protein binding proteins, antigen-binding fragments thereof, ADC, AEC or CAR according to the present invention bind to human ROR2, cynomolgus ROR2 and mouse ROR2 and compete for binding with an antibody containing variable domains from GK-5A1, MK-24C10 or MK-24F9, and preferably with an antibody containing variable domains from GK-5A1.

According to a preferred embodiment, the anti-ROR2 antibodies, antibody-based protein binding proteins, antigen-binding fragments thereof, ADC, AEC or CAR of the present invention bind to cynomolgus ROR2, murine ROR2 and human ROR2. According to a preferred embodiment, the anti-ROR2 antibodies, antibody-based protein binding proteins, antigen-binding fragments thereof, ADC, AEC or CAR according to the present invention bind to cynomolgus ROR2, mouse ROR2 and human ROR2 and contain CDRs from GK-5A1, MK -24C10 or MK-24F9 and preferably from GK-5A1. According to a preferred embodiment, the anti-ROR2 antibodies that bind antibody-based proteins, their antigen-binding fragments, ADC, AEC or CAR according to the present invention bind to cynomolgus ROR2, murine ROR2 and human ROR2 and contain variable domains from GK-5A1, MK-24C10 or MK-24F9 and preferably from GK-5A1.

According to a preferred embodiment, the anti-ROR2 antibodies, antibody-based protein-binding proteins, antigen-binding fragments thereof, ADC, AEC or CAR according to the present invention are mouse-derived. Anti-ROR2 antibodies that bind antibody-based proteins, antigen-binding fragments thereof, ADCs, AECs, or CARs of the present invention may exhibit advantageous lower aggregation propensity, improved manufacturability, and/or higher expression levels compared to fully human anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, ADCs, AECs, or CARs produced by methods involving phage display.

Also provided are polynucleotides encoding fully human anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof described herein, host cells transformed or transfected with these polynucleotides, and methods for producing various fully human anti-ROR2 antibodies, antibody-based binding proteins. , their antigen-binding fragments described herein. The present invention also provides substantially purified polynucleotides (DNA or RNA) that are identical or complementary to sequences encoding polypeptides containing segments or domains of the antibody chains or antigen-binding fragments described herein. When expressed from appropriate expression vectors, the polypeptides encoded by these polynucleotides are capable of exhibiting antigen-binding ability. Also provided in accordance with the present invention are polynucleotides that encode at least one CDR, and typically all three heavy or light chain CDRs, in fully human anti-ROR2 antibodies, antibody-based binding proteins, antigen-binding fragments thereof, described herein. Certain other polynucleotides encode all or substantially all of the heavy chain and/or light chain variable region sequence of the illustrated antibodies. Due to the degeneracy of the code, different nucleic acid sequences will code for each of the amino acid sequences of an immunoglobulin. The polynucleotides of the present invention can encode only the sequence of the variable region of the illustrated antibody. They can also encode both the variable region and the constant region of an antibody. Some of the nucleic acid polynucleotide sequences of the present invention encode a mature antibody heavy chain variable region sequence that is substantially identical (e.g., at least 80%, 90%, or 99%) to the represented antibody heavy chain variable region mature sequence. Certain other polynucleotide sequences encode a mature light chain variable region sequence that is substantially identical to the presented light chain variable region mature sequence. Some of the polynucleotide sequences encode a polypeptide that contains the variable regions of both the heavy chain and the light chain of one of the illustrated antibodies. Some other polynucleotides encode two polypeptide segments, which are respectively substantially identical to the heavy chain and light chain variable regions of one of the illustrated antibodies.

Polynucleotide sequences can be obtained by de novo solid phase DNA synthesis or by PCR mutagenesis of the existing sequence encoding the illustrated functional antibody. Direct chemical synthesis of nucleic acids can be carried out using methods known in the art, such as the phosphotriester method from Narang et al., Meth. Enzymol. 68:90, 1979; phosphodiester method from Brown et al., Meth. Enzymol. 68:109, 1979; diethylphosphoramidite method from Beaucage et al., Tetra. Lett. 22:1859, 1981; and the solid phase support method of US Pat. No. 4,458,066. The introduction of mutations into a polynucleotide sequence by PCR can be carried out as described, for example, in PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, mnis et al. (Ed.), Academic Press, San Diego, CA, 1990; Manila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991.

In accordance with the present invention, expression vectors and host cells are also provided for the production of fully human anti-ROR2 antibodies, antibody-based binding proteins, their antigen-binding fragments described herein. Various expression vectors can be used to express polynucleotides encoding functional antibody chains or binding fragments. Both viral and non-viral expression vectors can be used to generate antibodies in a mammalian host cell. Non-viral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for protein or RNA expression, and human artificial chromosomes (see, for example, Harrington et al., Nat. Genet. 15:345, 1997). For example, non-viral vectors useful for expressing antibody polynucleotides and polypeptides in mammalian (e.g., human) cells include pCEP4, pREP4, pThioHis A, B & C, pcDNA3.1/His, pEBVHis A, B & C (Invitrogen, San Diego, CA, USA), MPSV vectors, and numerous other vectors known in the art of expressing other proteins. Other suitable non-viral vectors include vectors containing any of Sleeping Beauty, PiggyBac and other transposon systems or sequences allowing transposition of vector components with said Sleeping Beauty, PiggyBac and other transposon systems. Suitable viral vectors include lentiviruses or other retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, SV40 vectors, papillomavirus, Epstein-Barr virus HBP, vaccinia virus, and Semliki forest virus (SFV) vectors. See, Brent et al., supra; Smith, Anna. Rev. microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68:143, 1992.

The choice of expression vector depends on the intended host cells in which the vector is to be expressed. Typically, expression vectors contain a promoter and other regulatory sequences (eg, enhancers) that are operably linked to polynucleotides encoding a functional chain or antigen-binding fragment of an antibody. In some embodiments, an inducible promoter is used to prevent the expression of the introduced sequences under conditions other than those for induction. Inducible promoters include, for example, tetracycline, arabinose, lacZ, metallothionein or heat shock protein promoters. Cultures of transformed or transfected host cells can be grown under non-inducing conditions without changing the population of coding sequences whose expression products are better tolerated by the host cells. For efficient expression of a functional chain or antibody fragment, other regulatory elements in addition to promoters may also be required or desirable. These elements typically include an ATG start codon and an adjacent ribosome binding site or other sequences. In addition, expression efficiency can be increased by incorporating enhancers suitable for the cell system employed (see, for example, Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol, 153 :516, 1987). For example, an SV40 enhancer or a CMV enhancer can be used to increase expression in mammalian host cells.

Expression vectors can also provide a position for a signal sequence for secretion to form a fusion protein with polypeptides encoded by the inserted functional antibody sequences. More commonly, the inserted functional antibody sequences are linked to signal sequences prior to incorporation into the vector. Vectors to be used to obtain sequences encoding functional antibody light and heavy chain variable domains sometimes also encode constant regions or portions thereof. Such vectors allow the expression of variable regions as fusion proteins with constant regions, thereby leading to the production of intact antibodies or fragments thereof. Typically, such constant regions are human.

Host cells for maintaining and expressing functional antibody chains can be either prokaryotic or eukaryotic. In accordance with some preferred embodiments, mammalian host cells are used to express and produce antibody polypeptides of the present invention. For example, they can be either a hybridoma cell line expressing endogenous immunoglobulin genes or a mammalian cell line carrying an exogenous expression vector. They include any normal mortal or normal or abnormal immortalized animal or human cell. In addition to the cells illustrated herein, a number of other suitable host cell lines capable of secreting intact immunoglobulins are also known in the art. These include, for example, CHO cell lines, various HEK 293 cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B-line cells, and hybridomas. The use of mammalian tissue culture for the expression of polypeptides is generally discussed in, for example, Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y., 1987. Expression vectors for mammalian host cells may include expression control sequences such as as an origin of replication, promoter and enhancer, as well as essential sites for messenger RNA processing such as ribosome binding sites, RNA splicing sites, polyadenylation sites and transcription terminator sequences. These expression vectors typically contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell type specific, developmental stage specific, and/or modulated or regulated. Suitable promoters include, without limitation, the EF1α and human UbC promoters illustrated herein, the metallothionein promoter, the constitutive adenovirus large late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter ( such as the human CMV immediate early promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

Methods for introducing expression vectors containing polynucleotide sequences of interest vary depending on the type of cellular host. For example, calcium chloride transformation or electroporation is often used on prokaryotic cells, while calcium phosphate treatment or electroporation can be used on other cell hosts (in general, see Sambrook et al., supra). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transfection, injection and microinjection, ballistic methods, virosomes, immunoliposomes, polycation:nucleic acid conjugates, deproteinized DNA, artificial virions, herpes virus structural protein VP22 fusion proteins( Elliot and O'Hare, Cell 88:223, 1997), agent-enhanced DNA uptake and ex vivo transduction. In the case of long-term production of high yield recombinant proteins, stable expression will often be desirable. For example, cell lines that stably express antibody chains or binding fragments can be generated using expression vectors of the present invention that contain viral origins or endogenous expression elements and a selectable marker gene. After the introduction of the vector, cells can be allowed to grow for 1-2 days in enriched medium before they are transferred to selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows the growth of cells that successfully express the introduced sequences in the selective medium. Proliferation of resistant transfected cells can be achieved using tissue culture techniques appropriate for the cell type.

Aspects of the present invention relating to the new ADC and AEC

The present invention also relates to antibody effector molecule (AEC) conjugates or antibody drug conjugates (ADC).

The term "antibody-effector molecule conjugates" (AEC) is used herein to describe liposomes or labeling agents associated with a fully human anti-ROR2 antibody, antibody-based binding protein, or antigen-binding fragment thereof, as described herein. The labeling agent enables the direct or indirect detection of the anti-ROR2 antibody or antigen-binding fragment to which it is attached. A liposome, as described in Bendas, BioDrugs, 15: 215-224, 2001, may allow controlled delivery of an agent to diseased cells. When preparing a liposomal conjugate, for example an immunoliposome, an agent, such as a chemotherapeutic or other drug, may be enclosed in a liposome for delivery to a target cell.

The term "antibody-drug conjugates" (ADC) is used in the context of this document to describe cytotoxic and/or cytostatic agents ("toxin") associated with a fully human anti-ROR2 antibody, antibody-based binding protein, or antigen-binding fragment, as described in this document. Cytotoxic and/or cytostatic agents are any agent known to inhibit growth and/or inhibit cell division and/or kill cells, especially malignant cells. Covalent conjugates of small molecular weight cytotoxic and/or cytostatic agents (molecular weight preferably <2500 Daltons) with antibodies or antigen-binding fragments specific for tumor cells are effective tools for specific targeting of cancer cells in order to destroy them.

In the ADC, the toxin payload can be conjugated to an antibody, an antibody-based binding protein, or an antibody fragment non-site-specifically via lysine or cysteine amino acid side chains using classical maleimide-functional chemical linkers or other chemicals known in the art. , which can mediate the conjugation of lysine or cysteine amino acid side chains. In the ADC of the present invention, the small molecular weight payload can also be site-specifically conjugated by a chemical, chemo-enzymatic or enzymatic conjugation mechanism known in the art, such as using bifunctional linkers, linkers enabling the Pictet-Spengler chemical reaction a on formyl-glycine to form enzyme-modified antibodies, with antibodies with a remodeled glycan component, or with bacterial transglutaminase or sortase enzymes.

According to one embodiment, the antibody-drug conjugate (ADC) has the general formula A-(L)n-(T)m, wherein

А представляет собой антитело, производное, модифицированный формат или фрагмент, которые описаны в данном документе,

A is an antibody, derivative, modified format, or fragment as described herein,

L представляет собой линкер,

L is a linker,

Т представляет собой полезную нагрузку в виде цитотоксического или цитостатического средства,

T is the payload in the form of a cytotoxic or cytostatic agent,

and in which n and m are integers from >1 to <10.

According to one embodiment, the antibody effector molecule (AEC) conjugate typically has the general formula A-(L)n-(T)m, wherein

А представляет собой антитело, производное, модифицированный формат или фрагмент по любому из п. 1,

A is an antibody, derivative, modified format or fragment according to any one of paragraph 1,

L представляет собой линкер,

L is a linker,

Т представляет собой метку, и в которой n и m представляют собой целые числа от >1 до <10.

T is a label, and in which n and m are integers from >1 to <10.

According to a preferred embodiment, the linker comprises or consists of at least one selected from the group consisting of: oligopeptide linker (including cleavable and non-cleavable oligopeptide linkers), hydrazine linker, thiourea linker, self-cleaving linker, succinimidyl-trans-4 -(maleimidylmethyl)cyclohexane-1-carboxylate (SMCC) linker, disulfide linker, thioether linker and/or maleimide linker.

The maleimide linker optionally contains cleavable spacers that can be cleaved by changes in pH, redox potential, and or specific intracellular enzymes.

One of skill in the art will appreciate that additional linkers may be suitable. Such linkers may be non-cleavable or may be cleaved by changes in pH, redox potential, or specific intracellular enzymes. Cleavable oligopeptide linkers include protease-cleavable linkers. It is understood that the linker may contain combinations of the foregoing. For example, the linker may be a valine-citrulline PAV linker.

According to one embodiment, the linker has at least one of the following amino acid sequences: - LPXTGn-, -LPXAGn-, -LPXSGn-, -LAXTGn-, -LPXTGn-, -LPXTAn- or -NPQTGn-, wherein Gn is an oligo - or polyglycine, where n is an integer from ≥1 to ≤21, An is an oligo- or polyalanine, while n is an integer from ≥1 to ≤21, and X is any possible amino acid sequence.

In accordance with a preferred embodiment, the linker is conjugated to the C-terminus of at least one subdomain of an antibody, antibody, derivative, modified format, or fragment.

According to one embodiment, prior to conjugation

антитело, производное, модифицированный формат или фрагмент несет мотив для распознавания сортазой, слитый или конъюгированный с С-концом по меньшей мере одного его субдомена, и

the antibody, derivative, modified format, or fragment carries a sortase recognition motif fused or conjugated to the C-terminus of at least one subdomain thereof, and

токсин или метка содержит глициновый отрезок длиной от ≥1 до ≤21 глициновых остатков, предпочтительно, длиной от ≥2 до ≤5 глициновых остатков.

the toxin or label contains a glycine segment ≥1 to ≤21 glycine residues in length, preferably ≥2 to ≤5 glycine residues in length.

The cytostatic agent in the ADC may be a plant, fungal or bacterial molecule. In some embodiments, the cytotoxic agent for conjugation to an antibody or antigen-binding fragment of the present invention is a small molecule cellular toxin, a peptide toxin, or a protein toxin. Many specific examples of these toxins are well known in the art. See, for example, Dyba et al., Curr. Pharm. Des. 10:2311-34, 2004; Kuyucak et al., Future Med. Chem. 6:1645-58, 2014; Beraud et al., Inflamm. Allergy Drug Targets. 10:322-42, 2011; and Middlebrook et al., Microbiol. Rev. 48:199-221, 1984. In some embodiments, the therapeutic agent is conjugated to an antibody. For example, the therapeutic agent may be a maytansinoid (eg, maytansinol or maytansinoid DM1), a taxane, calicheamicin, cematodin, monomethylauristatin (eg, monomethylauristatin E or monomethylauristatin F), pyrrolobenzodiazepine (PBD), indilino-benzodiazepine pseudodimer, or anthracycline. Therapeutic agents also include vincristine and prednisone. According to various embodiments, a therapeutic agent that can be used in the present invention may be an antimetabolite (eg, an antifolate such as methotrexate, a fluoropyrimidine such as 5-fluorouracil, a cytosine arabinoside, or a purine or adenosine analog); an intercalating agent (eg, an anthracycline such as doxorubicin, PNU-159682, daunomycin, epirabicin, idarubicin, mitomycin-C, dactinomycin or mithramycin, or other intercalating agents such as pyrrolobenzodiazepine); a DNA reactive agent such as calicheamicins, thiancimycins and other enediins; a platinum derivative (eg cisplatin or carboplatin); an acylating agent (eg nitrogen mustard, melphalan, chlorambucil, busulfan, cyclophosphamide, ifosfamide, nitrosoureas or thiotepa); an RNA polymerase inhibitor such as a-amanitin; an antimitotic agent (eg, a vinca alkaloid such as vincristine or a toxoid such as paclitaxel or docetaxel); a topoisomerase inhibitor (eg, etoposide, teniposide, amsacrine, topotecan); a cell cycle inhibitor (eg flavopiridol); or an agent that disrupts microtubule formation (eg, epothilone, tubulisin, pretubulisin, discodermolide analog, or eleutherobin analog). The therapeutic agent may be a proteasome inhibitor or a topoisomerase inhibitor such as bortezomib, amsacrine, etoposide, etoposide phosphate, teniposide or doxorubicin. Therapeutic radioactive isotopes include iodine ( ¹³¹ I), yttrium ( ⁹⁰ Y), lutetium ( ¹⁷⁷ Lu), actinium ( ²² 5Ac), praseodymium, astatine (At), rhenium (Re), bismuth (Bi or Bi) and rhodium (Rh). Anti-angiogenic agents include linomide, bevacizumab, angiostatin, and razoxane.

According to a preferred embodiment of the ADC, the cytotoxic or cytostatic payload is at least one selected from the group consisting of:

майтансиноидов,

maytansinoids,

ауристатинов,

auristatins,

антрациклинов,

anthracyclines,

калихеамицинов,

calicheamicins,

тубулизинов,

tubulizins,

дуокармицинов,

duocarmycins,

радиоизотопов,

radioisotopes,

липосом, содержащих полезную нагрузку в виде анатоксина,

liposomes containing a payload in the form of toxoid,

белковых токсинов,

protein toxins,

таксанов,

taxanes,

псевдодимеров индилино-бензодиазепина и/или

pseudodimers of indilino-benzodiazepine and/or

пирролобензодиазепинов, или представляет собой их производное.

pyrrolobenzodiazepines, or is a derivative thereof.

According to a preferred embodiment, the ADC toxin is selected from PNU-159682, which is described in Quintieri et al. (2005), and its derivatives, maytansine, monomethyl auristatin MMAE and monomethyl auristatin MMAF. According to a preferred embodiment, the ADC toxin attached to the linker, as shown by the wavy line, has the formula (i) as described in WO 2016/102679:

In accordance with the embodiment in which the toxin has the formula (i), it is preferred that the linker contains an alkyldiamino group of the form NH ₂ -(CH ₂ ) _m -NH ₂ where m ≥1 and ≤11, preferably m=2, whereby one amino group is directly bonded, as shown by the wavy line in formula (i), to form an amide bond. In addition, it is preferred that the second amino group is linked to an oligopeptide linker, which is more preferably an oligoglycine.

In some embodiments, the antibody-conjugating molecule is a protein (eg, an antibody) or an RNA or DNA aptamer.

In accordance with a preferred embodiment, the AEC label is at least one selected from the group consisting of: a fluorescent label (including a fluorescent dye or a fluorescent protein), a chromophore label, a radioisotope label containing iodine (for example, ¹²⁵ I), gallium ( ⁶⁷ Ga), indium ( ¹¹¹ I), technetium ( ^99m Tc), phosphorus ( ³² P), carbon ( ¹⁴ C), tritium ( ³ H), another radioactive isotope (for example, a radioactive ion), and / or protein a label such as avidin or streptavidin.

According to a preferred embodiment, ADC or AEC The ADC or AEC is characterized by a stoichiometric ratio between antibody and payload of any integer from ≥1 to ≤10, preferably 2 to 4. In the case of ADC, this ratio may also be referred to as the drug ratio and antibodies ("DAR"). In the case of a mixture or set of ADCs, the DAR may be a rational number representing the average of the individual DARs b as integers.

According to one embodiment of the ADC or AEC, only one or more of the heavy chains of the antibody or the corresponding antigen-binding fragment is conjugated to the payload.

According to one embodiment of the ADC or AEC, only one or more light chains of the antibody or the corresponding antigen-binding fragment are conjugated to the payload.

It is understood that a given ADC or AEC may simultaneously contain various associated toxins or labels; it is also possible for an anti-ROR2 antibody to be associated with a combination of both one or more labels and one or more toxins.

Antibodies or antigen-binding fragments of the present invention can be conjugated to a synthetic molecule using any type of suitable conjugation. Recombination-based genetic engineering techniques and incorporation of selenocysteine (eg, as described in US Pat. No. 8,916,159) can be used to conjugate the synthetic molecule. Other conjugation methods may include covalent linkage to native or engineered lysine side chain amines or cysteine side chain thiols. See, for example, Wu et al., Nat. Biotechnol, 23: 1 137-1 146 (2005). The synthetic molecule can be any molecule, such as one that targets a tumor.

In accordance with a preferred embodiment, ADC or AEC is produced using enzyme sortase-mediated site-specific antibody conjugation (SMAC technology), which is comprehensively disclosed in international application WO 2014140317, the rights of which belong to the same applicant. The contents of this publication are incorporated herein by reference. Sortases (also called transpeptidases-sortases) are from a group of prokaryotic enzymes that modify surface proteins by recognizing and cleaving a specific peptide motif called a "sortase recognition motif" or "sortase tag". Typically, a given sortase enzyme recognizes one or more sortase recognition motifs. Sortase enzymes may occur naturally or may have been genetically engineered (Dorr et al., 2014) and are used in SMAC technology to conjugate two proteins, one of which carries this recognition motif while the other carries an oligolicin peptide (Gly) _n . It is important to understand that in accordance with one particular embodiment (in which Streptococcus pyogenes sortase A is used, see below), oligoglycine (Gly) _n may optionally be replaced by oligoalanine (Ala) _n .

According to a preferred embodiment, ADC or AEC is produced by enzyme sortase-mediated site-specific conjugation:

a) an antibody or antigen-binding fragment as described herein that carries one or more sortase recognition motifs, and

b) one or more payload molecules bearing an oligoglycine tag;

or

a) an antibody or non-antigen binding fragment as described herein that bears one or more oligoglycine tags, and

b) one or more payload molecules carrying a motif for sortase recognition.

It is important to understand that in accordance with one particular embodiment (in which Streptococcus pyogenes sortase A is used, see below), oligoglycine (Gly) _n may optionally be replaced by oligoalanine (Ala) _n .

Preferably, the sortase recognition motif is fused or conjugated to the C-terminus of at least one antibody subdomain.

Preferably, the oligoglycine tag is ≥1 to ≤21 glycine residues in length, preferably ≥2 to ≤5 amino acids in length.

The present invention also relates to a process for producing AEC or ADC, the process comprising the following steps:

a) providing an antibody, derivative, modified format, or fragment as described herein, wherein the antibody or antigen-binding fragment carries one or more sortase recognition motifs,

b) providing one or more payload molecules bearing an oligoglycine tag, and

c) conjugation of the antibody or antigen-binding fragment and one or more payload molecules by sortase-mediated conjugation.

The term "oligoglycine tag" (also referred to as Gn or G(n)) refers to an oligoglycine with a length of n, which can be ≥1 to ≤21, preferably ≥1 to ≤5.

The present invention also relates to a process for producing AEC or ADC, the process comprising the following steps:

a) providing an anti-ROR2 antibody or antigen-binding fragment as described herein, wherein the antibody or antigen-binding fragment bears one or more oligoglycine tags,

b) providing one or more payload molecules carrying a motif for sortase recognition, and

c) conjugation of the antibody or antigen-binding fragment and one or more payload molecules by sortase-mediated conjugation.

According to yet another embodiment, said sortase enzyme recognition motif comprises at least one of the following amino acid sequences: LPXTG, LPXAG, LPXSG, LAXTG, LPXTA, or NPQTN, where X is any possible amino acid sequence.

According to yet another embodiment, the resulting linker has at least one of the following amino acid sequences: -LPXTGn-, -LPXAGn-, -LPXSGn-, -LAXTGn-, -LPXTGn-, -LPXTAn- or -NPQTGn-, wherein Gn is an oligo or polyglycine, where n is an integer from ≥1 to ≤21, An is an oligo or polyalanine, where n is an integer from ≥1 to ≤21, and X is any possible amino acid sequence.

Table 4 Sortase recognition motifs and resulting linkers

Genetically engineered sortases, including but not limited to the sortase A 2A-9 mutant and the sortase A 4S-9 mutant from Staphylococcus aureus, are described in Dorr et al. (2014), as well as the mutants described in Chen et al. (2011).

For reference and to illustrate the general idea of sortase-mediated transpeptidation, sortase A uses an oligoglycine stretch as a nucleophile to catalyze transpeptidation, in which the amino terminal of the oligoglycine produces a nucleophilic attack on the peptide bond connecting the last two C-terminal residues in the sortase tag. This results in cleavage of the peptide bond and formation of a new peptide bond between the second C-terminal residue in the sortase tag and the N-terminal glycine in the oligoglycine peptide, i. results in transpeptidation.

It is important to understand that in one particular embodiment (in which Streptococcus pyogenes sortase A is used, see above), oligoglycine (Gly)n may optionally be replaced with oligoalanine (A1a)n.

Before conjugation using sortase, the motif recognized by sortase may additionally carry other tags at its C-terminus, like His-tags, Mus-tags or Strep-tags (see Fig. 4a in international application WO 2014/140317, the content of which is incorporated herein). document by reference). However, because the peptide bond between the 4th and 5th amino acids in the sortase tag is cleaved during sortase A-mediated conjugation, these additional tags do not appear in the conjugated product.

A sortase tag, for example, can be fused to the C-terminus of a binding protein or a domain or subunit thereof by genetic fusion and co-expressed with it. According to another preferred embodiment, the sortase tag can be directly attached to the last naturally occurring C-terminal amino acid in immunoglobulin light chains or heavy chains, which in the case of the human immunoglobulin kappa light chain is the C-terminal cysteine residue, and which, in the case of a human IgG1 immunoglobulin heavy chain, may be the C-terminal lysine residue encoded by the human Fcγ1 cDNA. However, another preferred embodiment is also to directly attach the sortase tag to the second C-terminal glycine residue encoded by the human Fcγ1 cDNA, since typically terminal lysine residues in antibody heavy chains are excised during post-translational modification in mammalian cells. Thus, more than 90% of naturally occurring human IgG1 lack C-terminal lysine residues in IgG1 heavy chains.

Therefore, one preferred embodiment of the present invention is not to include C-terminal lysine amino acid residues in the human IgG1 heavy chain constant regions in Igγ1 heavy chain expression constructs labeled with a sortase recognition motif. Another preferred embodiment is to include C-terminal lysine amino acid residues in human IgG1 heavy chain constant regions in Igγ1 heavy chain expression constructs labeled with a sortase recognition motif.

According to yet another preferred embodiment, a sortase tag or an oligoglycine tag can be fused to the C-terminus of a human IgG1 heavy chain in which the C-terminal lysine residue encoded by the human Fcγ1 cDNA is replaced with an amino acid residue other than lysine, for preventing unproductive sortase reactions with the ε-amino group of the specified C-terminal lysine residue, leading to cross-linking of heavy chains with each other.

The present inventors have previously described that in some cases (e.g. at the C-terminus of Ig kappa light chains, see: Beerli et al. (2015) PloS One 10, e 131177) it is advantageous to add additional amino acids between the C-terminus binding protein and sortase tag. This has been shown to improve the efficiency of the sortase enzyme's "payload" conjugation to the binding protein. In the case of Ig kappa light chains, it was observed that adding 5 amino acids between the last C-terminal cysteine amino acid in the Ig kappa light chain and the pentapeptide motif for sortase improved conjugation kinetics, resulting in the C-terminals of Ig kappa light chains and heavy Ig chains can be conjugated with similar characteristics (see: Beerli et al. (2015) PloS One 10, e131177). Thus, another preferred embodiment is that optionally ≥1 and ≤11 amino acids are added between the last C-terminal amino acid in the binding protein or antibody subunit and the sortase tag.

According to a preferred embodiment, a G _n S peptide (where n is ≥1 to ≤21, preferably ≥1 to ≤5) is added between the last C-terminal amino acid in the binding protein or antibody subunit and the sortase tag.

Finally, according to yet another preferred embodiment, additional amino acids can be advantageously included between the C-terminus of the binding protein and the sortase tag or oligoglycine tag, which contain a sequence and/or a linker that can be cleaved by hydrolysis, by changing the pH, or by changing redox potential, or which can be cleaved by an enzyme other than sortase, for example by proteases.

In accordance with another aspect of the present invention, a chimeric ROR2 antigen receptor (CAR) is provided using an antibody, derivative, modified format or fragment as described above, fused or conjugated to at least one transmembrane region and at least one intracellular domain.

In addition, a cell is provided containing such a chimeric antigen receptor, the cell preferably being a genetically engineered T cell.

Also contemplated is the use of an antibody, derivative, modified format or fragment, antibody-drug conjugate or CAR or cell as described above to treat a patient who

страдает от неопластического заболевания,

suffering from neoplastic disease,

имеет риск развития неопластического заболевания, и/или

is at risk of developing neoplastic disease, and/or

у которого диагностировано неопластическое заболевание.

diagnosed with neoplastic disease.

Alternatively, a method is provided for treating a patient suffering from a neoplastic disease, at risk of developing a neoplastic disease, and/or diagnosed with a neoplastic disease, the method comprising administering one or more therapeutically active doses of an antibody, antibody-drug conjugate, or CAR, or cells as described above.

According to one embodiment, the neoplastic disease is at least one selected from the group consisting of

почечно-клеточной карциномы,

renal cell carcinoma,

остеосаркомы,

osteosarcomas,

рака почки.

kidney cancer.

In one aspect, a pharmaceutical composition is provided comprising an antibody, derivative, modified format or fragment, antibody-drug conjugate, or CAR, or cell as described above, together with one or more pharmaceutically acceptable ingredients.

According to yet another aspect, the present invention provides methods for killing or inhibiting the growth of a cell expressing ROR2 in vitro or in a patient, the method comprising administering to the cell a pharmaceutically effective amount or dose of an antibody, derivative, modified format or fragment, antibody drug conjugate, or CAR, or cells as described above, or a pharmaceutical composition as described above.

Preferably, the cell expressing ROR2 is a cancer cell.

The methods involve administering a therapeutically effective amount of an anti-ROR2 antibody, a binding protein based on the antibody, a fragment of the antibody, an ADC, or a CAR disclosed herein to a subject in need of treatment. This makes it possible to kill or inhibit the growth of a cell expressing ROR2 in a subject. According to various embodiments, the cell expressing ROR2 is a tumor cell. In accordance with a related aspect, the present invention provides methods for treating a disease or condition associated with increased ROR2 expression in a subject. These methods include administering a therapeutically effective amount of an anti-ROR2 antibody, an antibody-based binding protein, an antibody fragment, an ADC, or a CAR of the present invention to a subject who is afflicted with a disease or condition associated with overexpression of ROR2. This enables the treatment of a disease or condition in a subject. Some of these methods are directed to the treatment of cancer in a subject. Cancers that are susceptible to treatment with the methods of the present invention include, for example, neuroblastoma, sarcoma (and especially osteosarcoma), renal cell carcinoma, breast cancer, testicular cancer, ovarian cancer, pancreatic cancer. , kidney cancer, stomach cancer, prostate cancer, head and neck cancer, melanoma, squamous cell carcinoma, multiple myeloma and other cancers.

In various embodiments, the administered anti-ROR2 antibody, antibody-based binding protein, antibody fragment, or ADC or CAR based on them are F(ab)2, Fv, scFv, IgGACH2, F(ab')2, scFv2CH3, Fab, VL, VH, scFv4, scFv3, scFv2, dsFv, Fv, (scFv)2, or synthetic IgG. In some embodiments, the administered anti-ROR2 antibody, antibody-based binding protein, or fragment of the antibody is conjugated to a synthetic molecule. According to some of these embodiments, an anti-ROR2 antibody-based protein binding protein fragment of the antibody is conjugated to the transmembrane region and the T cell receptor (TCR) intracellular signaling domain to form a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor (CAR) is present on the T cell to be administered to the subject. In some other embodiments, the antibody may be conjugated to a cytotoxic agent, a radioactive isotope, or a liposome.

The terms "treatment" or "treatment" as used in the context of this document do not necessarily imply 100% or complete cure. More specifically, there are variable levels of treatment known to those skilled in the art to have a potential beneficial effect or therapeutic effect. In this regard, the method according to the present invention can provide any amount of any level of treatment. Moreover, the treatment provided by the method of the present invention may include the treatment of one or more conditions or symptoms of the disease being treated. In particular, the treatment can be carried out as an intravenous infusion.

In one embodiment, a fully human anti-ROR2 antibody, an antibody-based binding protein or antibody fragment, a bi- or multispecific antibody, an ADC or a CAR is administered as monotherapy. In an alternative embodiment, a fully human anti-ROR2 antibody, antibody-based binding protein or antibody fragment, bi- or multispecific antibody, ADC or CAR is administered concomitantly or concurrently or sequentially with additional therapeutic agents.

In particular, a fully human anti-ROR2 antibody, an antibody-based binding protein or antibody fragment, a bi- or multispecific antibody, an ADC, or a CAR may be administered at a dose of about 0.1-20 mg/kg.

The term "subject" refers to humans and non-human animals (especially non-human mammals) and preferably humans. In addition to binding to ROR2 of an antibody, a binding protein based on an antibody or a fragment of this antibody, a bi- or multispecific antibody, ADC or CAR, they can also bind to ROR2 from other species, making them effective for the treatment of these species.

The present invention also relates to a method of treating a subject suffering from, at risk of developing and/or diagnosed with a neoplastic disease, comprising administering a fully human anti-ROR2 antibody, a binding protein based on an antibody or a fragment of this antibody, ADC or CAR, as described in this document.

In particular, said neoplastic disease is selected from the group consisting of: neuroblastoma, sarcoma (and especially osteosarcoma), renal cell carcinoma, breast cancer, testicular cancer, ovarian cancer, pancreatic cancer, kidney cancer, gastric cancer , prostate cancer, head and neck cancer, melanoma, squamous cell carcinoma, multiple myeloma and other cancers.

In accordance with certain related aspects, the present invention provides pharmaceutical compositions or kits that comprise a therapeutically effective amount of a fully human anti-ROR2 antibody or an antibody, a binding protein based on an antibody or antibody fragment, a bi- or multispecific antibody, or an ADC described herein, and a pharmaceutically acceptable carrier. Some kits according to the present invention may additionally contain one or more immunoassay buffers.

According to some embodiments, the compositions of the present invention comprise a carrier, preferably a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier may be one or more compatible solid or liquid excipients, diluents, other excipients, or encapsulating agents that are suitable for administration to a human or subject receiving veterinary care (e.g., a physiologically acceptable carrier or a pharmacologically acceptable carrier). The term "carrier" defines an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the use of the active ingredient, for example, the administration of the active ingredient to a subject. A pharmaceutically acceptable carrier may be mixed with one or more of the active ingredients, for example a fusion molecule, and with each other if more than one pharmaceutically acceptable carrier is present in the composition, such that it does not substantially impair the desired pharmaceutical efficacy. Pharmaceutically acceptable materials can generally be administered to a subject, such as a patient, without causing significant undesirable physiological effects such as nausea, dizziness, rash, or stomach upset. For example, it is desirable that a composition containing a pharmaceutically acceptable carrier is not immunogenic when administered to a human patient for therapeutic purposes.

The pharmaceutical compositions of the present invention may further contain suitable buffering agents, including, for example, acetic acid in the salt, citric acid in the salt, boric acid in the salt, and phosphoric acid in the salt. The compositions may also optionally contain suitable preservatives such as benzalkonium chloride, chlorobutanol, parabens and thimerosal. The pharmaceutical compositions of the present invention may be present in unit dosage form and may be prepared by any suitable method, many of which are well known in the pharmaceutical art. Such methods include the step of combining the antibody or antigen-binding fragment of the present invention with the carrier which constitutes one or more additional ingredients. Typically, the composition is prepared by uniformly and intimately bringing the active agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

A composition suitable for parenteral administration suitably comprises a sterile aqueous formulation of the composition of the present invention which is preferably isotonic with the recipient's blood. This aqueous formulation can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic, parenteral acceptable diluent or solvent, such as a solution in 1,3-butanediol. Among the acceptable media and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fatty oils are commonly used as a solvent or suspending medium. Any light fixed oil can be used for this purpose, such as synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injection solutions. Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular and other administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.

The preparation of pharmaceutical compositions according to the present invention, as well as their administration in various ways, can be carried out in accordance with methods well known in the prior art. See, for example, Remington: The Science and Practice of Pharmacy, Mack Publishing Co., ^20th ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978. Delivery systems useful in the context of the present invention include slow release, sustained release, and sustained release delivery systems, whereby the delivery of the composition according to the present invention occurs before sensitization of the site to be treated, and for a sufficient period of time to cause sensitization. The composition according to the present invention can be used in conjunction with other therapeutic agents or therapies. Such systems may avoid repeated administrations of the composition of the present invention, thereby increasing convenience for the subject and the clinician, and may be particularly suitable for certain compositions of the present invention.

Many types of release delivery systems are available and known to those skilled in the art. Suitable release delivery systems include those based on polymers such as poly(lactide-glycolide), oxalate copolymers, polycaprolactones, polyetheramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Drug-containing microcapsules of the above polymers are described, for example, in US Pat. No. 5,075,109. Delivery systems also include non-polymeric systems that are lipids, including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-, di-, and triglycerides; hydrogel release systems; systems based on sylastic material; peptide based systems; wax coatings; pressed tablets using traditional binders and excipients; partially fused implants, etc. Specific examples include, without limitation: (a) erosion systems in which the active composition is contained in a form within a matrix, such as those described in US Pat. and (b) diffusion systems in which the active ingredient permeates at a controlled rate from a polymer such as that described in US Pat. Nos. 3,832,253 and 3,854,480. In addition, pump-based hardware delivery systems, some of which are adapted for implantation, can be used.

The present invention also provides kits suitable for carrying out the methods of the present invention. As a rule, kits contain two or more components required for the implementation of therapeutic or diagnostic methods according to the present invention. Kit components include, without limitation, one or more antibodies or antigen-binding fragments of the present invention, appropriate reagents, and/or equipment. In some embodiments, kits may contain an antibody or antigen-binding fragment of the present invention and an immunoassay buffer suitable for ROR2 detection (eg, by ELISA, flow cytometry, magnetic sorting, or FACS). The kit may also contain one or more microtiter plates, standards, assay diluents, wash buffers, adhesive plate caps, magnetic beads, magnets, and/or instructions for carrying out the method of the present invention using the kit. The kit may include an antibody or antigen-binding fragment of the present invention associated with a support (eg, a multiwell plate or chip) that is suitably packaged and suitable for ROR2 detection. In some embodiments, the kit includes an antibody or antigen-binding fragment of the present invention that is conjugated to a label, such as a fluorescent label, a biologically active enzyme label, a luminescent label, or a chromophore label. The kits may further include reagents for visualizing the conjugated antibody, such as an enzyme substrate. In some embodiments, kits include an antibody or antigen-binding fragment of the present invention that is conjugated to a contrast agent, and optionally one or more reagents or equipment assemblies suitable for imaging the antibody in a subject.

Typically, an antibody or antigen binding fragment of the present invention in a kit is suitably packaged, for example, in a vial, bag, ampoule, and/or any container suitable for a therapeutic or detection method. The components of the kit may be provided as concentrates (including lyophilized compositions) which may be further diluted prior to use, or they may be provided in a concentration for use. In the case of using an antibody, derivative, modified format or fragment according to the present invention in vivo, single doses can be provided in sterilized containers containing the desired amount and concentration of the components.

Aspects of the present invention relating to methods for detecting

In accordance with yet another aspect of the present invention, a method is provided for determining whether

страдает ли предполагаемый пациент от неопластического заболевания или иммунного заболевания,

whether the intended patient suffers from a neoplastic disease or an immune disease,

имеет ли предполагаемый пациент риск развития неопластического заболевания или иммунного заболевания, и/или

whether the intended patient is at risk of developing a neoplastic disease or an immune disease, and/or

диагностировано ли у предполагаемого пациента неопластическое заболевание или иммунное заболевание,

whether the intended patient has been diagnosed with a neoplastic disease or an immune disease,

said method comprising treating a sample taken from the subject with an antibody-effector molecule conjugate as described above.

In one embodiment, the neoplastic disease or immune disease is suitable for treatment with an anti-ROR2 antibody, an anti-ROR2 antibody-drug conjugate, or a CAR T cell containing an anti-ROR2 antibody.

In accordance with some other embodiments, the present invention relates to a method for detecting an altered level of ROR2 (e.g., cell surface ROR2) in a biological sample, for example, compared to a control using any of FACS, immunohistochemistry (IHC), or Western blotting. Typically, the method includes contacting a biological sample with an antibody, an antibody-based binding protein, a fragment of that antibody according to the present invention, and determining the amount of antibody that selectively binds to material (e.g., cells) in the sample, to thereby determine the level of ROR2 in the biological sample. The biological sample can be obtained from a cell culture or from the subject under investigation, for example, blood plasma or a tissue sample from a subject who has a disease or condition associated with an elevated ROR2 level in the subject, is suspected to have a disease or condition associated with an elevated ROR2 level in the subject , or is at risk of developing a disease or condition associated with elevated ROR2 levels in the subject. The control level desirably corresponds to the level of ROR2 detected using the same antibody in the corresponding sample(s) from one or more control cultures or from subjects not suffering from the disease. Methods of using an antibody of the present invention to determine ROR2 levels may include any immunological assay, such as immuno-(western) blotting, enzyme-linked immunosorbent assay (ELISA), immunohistochemical assay (IHC), and flow cytometry, such as fluorescence-activated assay. cell sorting (FACS).

Detection methods can be used to screen for the presence of a disorder associated with elevated ROR2. The methods include obtaining a sample from an investigational subject in need of screening, e.g., a subject who has an elevated ROR2 disorder, is suspected to have an elevated ROR2 disorder, or is at risk of developing an elevated ROR2 disorder. . The level of ROR2 (eg, amount or concentration) in a sample is measured using an antibody binding protein based on an antibody, a fragment of this antibody according to the present invention, and the level in the sample is compared with a control level of ROR2. The control level is, for example, the average level (eg, amount or concentration) in the sample(s) from one or preferably more subjects in the control group that do not have a disorder associated with an elevated ROR2 level. Alternatively, the control level may correspond to the level or mean level of ROR2 in one or more samples taken from the study subject one or more times prior to the study, such as when the study subject did not have a condition associated with an elevated ROR2 level, or he did not manifested a condition associated with an increased level of ROR2. A significantly higher level of ROR2 in the biological sample compared to the control level indicates a disorder associated with an elevated level of ROR2 in the subject. In subjects such as humans, in cases where cell surface expression of ROR2 is predominantly restricted to embryonic development, the control level of ROR2 may be zero or absent. Thus, in accordance with some embodiments of the detection method of the present invention, any significant and detectable amount of ROR2 in a biological sample may be indicative of a disorder associated with an elevated ROR2 level in a subject.

In addition, detection methods can be used to monitor the development of a disorder associated with an elevated ROR2 level. The method includes obtaining a sample from a subject in need of screening, such as a subject who has been diagnosed with an elevated ROR2 disorder or is suspected of having a disorder associated with an elevated ROR2 level. The level of ROR2 in a sample is measured using an antibody binding protein based on an antibody, a fragment of this antibody according to the present invention, and the level in the sample is compared with a control level corresponding to the level or average level of ROR2 in one or more samples taken from the test subject one or more times before research. ROR2 levels that are significantly increased or decreased compared to controls indicate that the subject's disorder is worsening or improving, respectively. The above detection methods can be used to screen for the presence or to monitor the development of disorders, including, for example, CLL, ALL, mantle cell lymphoma, neuroblastoma, sarcoma, renal cell carcinoma, breast cancer, lung cancer, colon cancer. colon, head and neck cancer, melanoma and other cancers.

In accordance with some embodiments, the present invention provides methods for screening a subject for an altered ROR2 level. Typically, the methods involve administering to a subject an antibody, an antibody-based binding protein, a fragment of the antibody of the present invention, which is conjugated to a label (e.g., a contrast agent), visualizing the subject in a manner suitable for detecting the label, and determining whether the area in the subject has an altered the density or concentration of the label compared to the background level of the label in the surrounding tissue. Alternatively, the methods include determining if an altered density or concentration of a label is present in an area compared to the density or concentration of a label previously detected in the same area of the subject. Methods for imaging a subject may include radiographic imaging, computed tomography (CT) imaging (e.g., CT angiography (CT) imaging), magnetic resonance (MR) imaging, magnetic resonance angiography (MRA), imaging using radiological medical techniques, ultrasound (US) imaging, optical imaging, elastography, infrared imaging, microwave imaging, and the like, which are suitable for detecting a label conjugated to an antibody. According to a preferred embodiment, the subject has, is suspected of having or is at risk of developing a ROR2 expressing tumor such as CLL, ALL, mantle cell lymphoma, neuroblastoma, sarcoma, renal cell carcinoma, breast cancer, lung cancer, colon cancer , head and neck cancer, melanoma and other cancers, and the method is used to screen for the presence of a tumor or detect the presence of a tumor. According to yet another embodiment, the method can be used to monitor the size or density of an ROR2-expressing tumor over time, such as during a course of treatment.

Thus, the present invention relates to methods for detecting an altered level of ROR2 in a subject. The methods include (a) obtaining a biological sample from a subject, (b) contacting the sample with an anti-ROR2 antibody, antibody-based binding protein, antibody fragment, as disclosed herein, (c) determining the level of ROR2 in the biological sample, and (d ) comparison of the level of ROR2 in the biological sample with the control level of ROR2. This makes it possible to determine if the ROR2 level in the biological sample is altered from a control ROR2 level. In these methods, an elevated ROR2 level in a subject compared to a control level is indicative of a disease or condition associated with increased expression of ROR2 in the subject. For example, detection of increased expression of ROR2 may indicate the presence of neuroblastoma, osteosarcoma, renal cell carcinoma, breast cancer, gastric cancer, prostate cancer, melanoma, squamous cell carcinoma, or multiple myeloma in the subject.

In accordance with yet another embodiment, the present invention relates to methods for detecting an ROR2-expressing tumor in a subject. These methods involve (a) administering an anti-ROR2 antibody, an antibody-based binding protein, a fragment of the antibody according to the present invention to a subject who has, is suspected to have, or is at risk of developing an ROR2-expressing tumor, and (b) imaging the subject to identify an area of altered density or the concentration of the conjugated label, wherein the density or concentration is relative to (i) background in a nearby tissue or (ii) a density or concentration previously detected in the same area in the subject. The existence of an area of altered density or concentration of the conjugated label indicates the presence of a ROR2-expressing tumor in the subject.

Aspects of the present invention relating to cells and T cells

In accordance with some embodiments, the present invention provides methods of treating a subject who has a disorder associated with elevated ROR2 levels, is suspected to have a disorder associated with elevated ROR2 levels, or is at risk of developing a disorder associated with elevated ROR2 levels, using adoptive transfer of subjected exposure to the genetic engineering techniques of T cells described herein that express an antibody or antigen-binding fragment of the present invention as a chimeric antigen receptor (CAR) that selectively binds ROR2. Recombinant DNA technology can be used to introduce CAR-encoding genetic material into any suitable T cells, such as central memory T cells, from a subject to be treated. T cells carrying genetic material can be expanded (eg, in the presence of cytokines). The genetically engineered T cells are transferred to the patient, typically by infusion. The transferred T cells of the present invention can then elicit an immune response against ROR2-expressing cells in a subject. The adoptive transfer method can be used, for example, to treat subjects who have or are suspected of having any of the cancers associated with ROR2, including CLL, ALL, mantle cell lymphoma, neuroblastoma, sarcoma, renal cell carcinoma, breast cancer. cancer, lung cancer, colon cancer, head and neck cancer, melanoma and other types of cancer. In accordance with some embodiments, the above methods of treatment may further include co-administration of a second therapeutic agent for treating a disorder associated with elevated ROR2. For example, if the disorder being treated includes a ROR2-expressing cancer, the method may further include co-administration of a cytotoxic, cytostatic, or anti-angiogenic, or immunostimulatory agent (e.g., immune checkpoint inhibitor antibodies, e.g., but not limited to, antibodies that bind with PD1, PDL1, CTLA4, OX40, TIM3, GITR, LAG3, etc.), suitable for cancer treatment. If the cancer is a B-cell malignancy, the method may further include, for example, co-administration of rituximab, alemtuzumab, ofatumumab, ocrelizumab, or a CHOP chemotherapy regimen.

Thus, the present invention further provides eukaryotic or non-eukaryotic cells (e.g., T-lymphocytes) that have been genetically engineered using recombinant DNA technology to produce fully human anti-ROR2 antibodies or antigen-binding fragments of the present invention. Eukaryotic or non-eukaryotic cells can be used as expression systems for producing an antibody of the present invention. In accordance with some embodiments, the present invention provides ROR2-targeted immune cells that have been genetically engineered to recombinantly express a ROR2-specific non-human or humanized antibody or antigen-binding fragment of the present invention. For example, the present invention provides a T cell that has been genetically engineered to express a human anti-ROR2 antibody or antigen-binding fragment of the present invention (e.g., scFv, scFv-Fc, or (scFv)2) that is linked to a synthetic molecule containing one or several of the following domains: a spacer or hinge region (eg, a CD28 sequence or an IgG4 hinge-Fc sequence), a transmembrane region (eg, a canonical transmembrane domain), and an intracellular T-cell receptor (TCR) signaling domain, thus forming a chimeric antigen receptor (CAR) or T-cell body. Intracellular TCR signaling domains that may be included in the CAR (or T cell body) include, but are not limited to, the CD3ξ, FcR-γ, and Syk-PT signaling domains, as well as the co-signaling domains of CD28, 4-1 BB and CD134. Methods for constructing CAR-expressing T cells (or T cell bodies) are known in the art. See, for example, Marcu-Malina et al., Expert Opinion on Biological Therapy, Vol. 9, No. 5 (posted online April 16, 2009).

EXAMPLES

Although the present invention has been illustrated and described in detail in the description of the drawings and the foregoing description, such illustration and description are considered illustrative or exemplary and not restrictive; the present invention is not limited to the disclosed embodiments. Other variants of the disclosed embodiments may be understood by those skilled in the art and carried out by them in the practical implementation of the claimed invention as a result of studying the drawings, the disclosure and the attached claims. In the claims, the word "comprising" does not exclude other elements or steps, and the singular forms do not exclude the plural. The fact that certain measurands are listed in different dependent claims does not in itself indicate that a combination of these measurands cannot be used to advantage. Any reference positions in the claims should not be construed as limiting the scope.

All amino acid sequences disclosed herein are in N-terminal to C-terminal orientation; all nucleic acid sequences disclosed herein are in 5' to 3' orientation.

Example 1 Creation and Screening of an Antibody Library (Figure 1)

Expression of the hROR2-TwinStrep antigen. The EBNA expression vector pCB14b-hROR2-Thr-ECD-TwinStrep (where Thr is threonine) driving the expression of the hROR2 extracellular domain (ECD) labeled at the C-terminus with the TwinStrep tag was transfected into HEK293T using Lipofectamine® LTX with PLUS® reagent ( Thermo Fisher Scientific, 15388100). After 1 day incubation (37° C., 5% CO ₂ , growth medium: Dulbecco's Modified Eagle's Medium (DMEM) high glucose (4.5 g/l), with L-glutamine at 10% (v/v) fetal calf serum (FCS), 100 IU/ml penicillin-streptomycin-fungisone and 2 mM L-glutamine (all from Bioconcept)) cells were expanded under selection conditions (2 μg/ml puromycin (Sigma-Aldrich, P8833-25 mg stock solution at a concentration of 2 mg / ml)). Cells were separated and further expanded (37°C, 5% CO ₂ ); upon reaching confluence, hyperflasks vials were coated with 20 μg/ml poly-L-lysine (Sigma-Aldrich, P1524) for 2 hours at 37° C. and washed twice with PBS. Cells were then trypsinized, washed with PBS, and plated on poly-L-lysine-coated hyperflasks. Once confluent again, cells were washed with PBS followed by medium exchange using production medium (DMEM/F-12, Gibco/Thermo Fisher Scientific, 31330-038) supplemented with 1 mg/mL puromycin (Sigma-Aldrich, P8833), 100 IU /ml penicillin-streptomycin-fungisone (Bioconcept, 4-02F00-H), 161 µg/ml N-acetyl-L-cysteine (Sigma-Aldrich, A8199) and 10 µg/ml reduced L-glutathione (Sigma-Aldrich , G6529). The supernatant, which was collected biweekly and filtered (0.22 μm) to remove cells, was stored at 4°C until purification. The removed supernatant was replaced with fresh production medium. Rapid analysis of all collected supernatants by polyacrylamide gel electrophoresis confirmed the presence of bands corresponding to hROR2.

For purification, the filtered supernatant was loaded onto a column suitable for strep binding; purification and elution were performed according to manufacturer's protocols on an AEKTA pure system (GE Healthcare). Fractions were analyzed for protein purity and integrity by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Protein-containing fractions were mixed and buffer exchanged using Amicon filter units (Millipore, Schaffhausen, CH, UFC901008) to a dilution ratio of ≥1:100 in PBS and then filter sterilized using a low retention filter (0.20 µm, Carl Roth, Karlsruhe, Germany, PA49.1).

Immunization of H2L2 mice with hROR2-TwinStrep. Each of the five transgenic humanized H2L2 mice (obtained from Harbor Biomed; H2L2 mice were the result of crossing the following lines of mice: F129, fvb/n and C57BL6, and when immunized they produce antibodies with a human variable domain and rat constant domain disclosed in international application WO 2010/070263 A1) aged 6-10 weeks were immunized with two intraperitoneal injections (IP) followed by one intravenous injection of 100 μl hROR2-TwinStrep (from Example 1) formulated according to Table 5.

Table 5. Immunization regimen for H2L2 mice

Blood samples were taken from each mouse from the tail vein on Days: -7, 7 and 28 relative to the first injection and by heart puncture on Day 49. All procedures described in this document, which involved animals, were consistent with the Swiss guidelines for care and keeping animals.

Determination of antibody titers. The collected blood samples were incubated at room temperature for 15-60 minutes to allow the blood to clot and then centrifuged to obtain serum in which antibody titers were assessed. To do this, ELISA plates were coated with 100 µl of 2 µg/ml hROR2-TwinStrep (from example 1) in sodium bicarbonate coating buffer (0.1 M Na ₂ CO ₃ , 0.1 M NaHCO ₃ , pH 9.6 ). After incubation for 1 day at 4°C, the plates were washed with PBS supplemented with 0.05% (v/v) Tween 20 and blocked with 150 μl of PBS supplemented with 0.05% (v/v) Tween 20 and 3% bovine serum albumin (BSA). Serum samples were diluted 100-fold and a 2.5-fold dilution series was prepared from them by adding 50 μl of each diluted sample to the appropriate ELISA plates. After 1 hour incubation at 37° C., the plates were washed with PBS supplemented with 0.05% (v/v) Tween 20 before adding an HRP-conjugated anti-rat FC-gamma fragment antibody (Jackson Immunoresearch, 112-036-071). After additional washing of the plate, the plates were washed with 50 μl of Sigmafast OPD Tablet set (Sigma, P9187) and the reaction was stopped by adding 2 M sulfuric acid. The plates were read on an ELISA plate reader (OD 490) and the results are shown in FIG. 2.

MACS isolation of hROR2 specific B cells from H2L2 mice. Mice were sacrificed 49 days after the initial antigen injection and mouse spleens were harvested. The spleens were transferred to a gentleMACS C-tube (cat. no. 130-093-237) containing 2.4 ml RPMI-10%FCS and homogenized using a gentleMACS Octo Dissociator (Miltenyi Biotec) before being filtered through a cell sieve (tubes for FACS, BD Flacon, 734-0001) to produce single cell suspensions.

hROR2-specific B cells from mouse splenocytes were then selected by MACS sorting: B cells were first isolated using the Pan B Cell Isolation Kit, human (Miltenyi Biotec, 130-095-813) by negative selection. For this, splenocyte cells were washed and suspended in MACS buffer (2-4 x 106 cells in 600 µl) before adding 2 µl of mouse IgG (ChromPure, Jackson Immunoresearch, catalog # 015-000-003 at a concentration of 20 µg/ml), incubating on ice for 15 minutes. Cells were washed and resuspended in 40 µl cold MACS buffer. Cells other than B cells were magnetically labeled by adding 10 µl of a biotin labeled pan-B cell antibody cocktail at 4° C. followed by the addition of 30 µl of cold MACS buffer and 20 µl of anti-biotin antibody microbeads. The final volume was adjusted to 500 μl with cold MACS buffer.

Cells other than B cells were removed by passing the cell suspension through an LD column (Miltenyi Biotec, 130-042-901) in a magnetic field using a QuadroMACS™ separator (Miltenyi Biotec, 130-091-051) and collecting the eluate.

In parallel, hROR2-TwinStrep (12 μg, from example 1) was incubated with Strep-Tactin® magnetic nanobeads (IBA 6-5500-005, 50 μl at 7.044 mg/ml) and MACS buffer at 4°C and loaded onto LS column (Miltenyi Biotec, 130-042-401) in a magnetic field to wash out unbound antigen. The antigen loaded beads were then resuspended with an LD column eluate (containing B cells) and incubated for 45 minutes on ice. After centrifugation and washing with MACS buffer, the mixture was placed on an LS column, washing in a magnetic field. Cells that subsequently washed out with the beads outside of the magnetic field are antigen-positive B cells.

Creation of an antibody cDNA library. Antigen positive B cells were resuspended in 500 µl of TRI reagent; 100 μl of chloroform was added after a 5 minute incubation. After vortexing and centrifugation, the clear upper phase was transferred to an Eppendorf tube, to which 1 µl of glycogen and 250 µl of isopropanol were added. After mixing, incubation, washing and centrifugation, the supernatant was removed by decantation and 500 μl of ice-cold ethanol (75%) was added to the RNA pellet with stirring by inversion. The RNA pellet was then centrifuged and the supernatant decanted before air drying.

The RNA was reverse transcribed into cDNA using standard techniques. The variable domains were amplified on a PCR cycler using human primers in two steps: the first set of primers according to Table 6, (a) the extended 5' end of the variable domain to include the upstream nucleotide sequence corresponding to the portion of the leader peptide and (b) the extended 5 '-end of the rat constant domain. The second set of primers included: (a) to complete the 5'-end, nucleotide sequences that provide the possibility of obtaining a complete leader peptide, as well as a NotI restriction site; (b) to amplify the fully human variable domain from the 3' end using a nucleotide sequence binding to the 3' end of the J domain, as well as Nhel or BsiWI restriction sites for IgG and IgM or IgK, respectively.

Table 6. Primers used to create the cDNA library.

Transposition-mediated creation of cell libraries and characterization of libraries. Variable domain-encoding cDNA antibodies were then cloned into vectors with encoded restriction sites next to nucleotides encoding human constant domains and between nucleotides encoding sequences functional with PiggyBac transposase as well as ampicillin resistance. The library was characterized using Miniprep followed by sequencing. The library consisted of 6×10 ⁶ to 5×10 ⁷ members.

They were co-transfected into 63-12 mouse A-MuLV-transformed pre-B cells with a second vector encoding PiggyBac transposase for transposition-mediated B cell surface display and antibody secretion (“Transpo-mAb Display”) as described in Waldmeier. et al., 2016.

Functional screening of supernatants from clones and selection of clones. B cells were single cells sorted by FACS using FACSAria and were selected based on positive double staining with anti-Strep tag antibody (IBA, 2-1555-050) and polyclonal PE-conjugated anti-IgG antibody (ebioscience, 12- 4998-82); up to 288 clones per IgG/IgK and IgM/IgK library per mouse were selected and grown in SF-IMDM medium with 2% (v/v) fetal bovine serum (FCS), 100 IU/ml penicillin-streptomycin-fungisone, 50 μM beta -mercaptoethanol and 2 mM L-glutamine (all from Bioconcept, Allschwil, Switzerland)) at 37° C. and 7.5% CO ₂ (FIG. 3). Antibodies in supernatants from selected B cell clones were then assessed by two means: (1) for binding to hROR2 by ELISA and (2) for mediating cytolysis of hROR2 overexpressing EMT-6 cells after binding to hROR2 using secondary ADC. binding to antibodies to ROR2.

Binding of hROR2 by Single Spot ELISA Half of the wells in 96-well plates were coated with 50 μl of 2 μg/ml anti-human Fc antibody (Jackson Immunoresearch, 109-006-008) and the other half were coated with 50 μl of 2 μg/ml hROR2-Twin- Strep (from example 1), both in wash buffer (0.1 M Na ₂ CO ₃ , 0.1 M NaHCO ₃ , pH 9.6), and stored overnight at 4°C. After blocking each well with 150 µl of PBS supplemented with 0.05% (v/v) Tween 20 and 3% (w/v) bovine serum albumin (BS A) for 1 hour at 37°C per well 50 μl of diluted supernatants (diluted 5-fold in PBS supplemented with 1% (w/v) bovine serum albumin (BSA) and 0.05% (v/v) Tween 20) were added. For comparison, serially diluted 1:20 samples of purified anti-ROR2 antibody as well as isotype control antibodies were added to the plate, starting at 0.5 μg/ml in PBS supplemented with 1% (w/v) bovine serum albumin (BSA) and 0. 05% (v/v) Tween 20. After 1 hour incubation at 37°C, plates were washed with PBS supplemented with 0.05% (v/v) Tween 20 before adding HRP-conjugated anti-human IgG antibody (Jackson Immunoresearch, 109 -036-008). After additional washing of the plate, the plates were washed with 50 μl of Sigmafast OPD Tablet set (Sigma, P9187) and the reaction was stopped by adding 2 M sulfuric acid. The plates were read on an ELISA plate reader (OD 490).

Single Spot Secondary Antibody Cytolysis Assay: EMT6 cells genetically engineered to overexpress hROR2 from Example 7 were plated at 1000 cells in 100 µl per well in complete DMEM (Dulbecco's Modified Eagle's Medium (DMEM) high glucose (4.5 g/l), with L-glutamine with 10% (v/v) fetal bovine serum (FCS), 100 IU/ml penicillin-streptomycin-fungisone and 2 mM L-glutamine (all from Bioconcept, Alschwil , Switzerland)) and incubated at 37°C and 5% CO ₂ . The following day, 15 µl of the undiluted supernatant or 15 µl of a serially diluted 2-fold sample of purified anti-ROR2 antibody, starting at a concentration of 10 µg/ml, was added to the cells. After incubation for 30 minutes, 35 μl of anti-hu-IgG-CL-PNU antibody (Moradec, AH-102PN-50) was added at a concentration of 0.32 μg/ml. After an additional three days, the plates were removed from the incubator and equilibrated at room temperature. After about 30 minutes, 50 μl was removed from each well, and then 50 μl of CellTiter-Glo® 2.0 luminescent solution (Promega, G9243) was added to each well. After shaking the plates at 750 rpm. for 5 minutes followed by a 20 minute incubation without shaking, luminescence was measured on a Spark 10M plate reader with an integration time of 1 second per well.

Antibodies showing a high level of binding to ROR2, as well as low cell viability, thus indicating them as good candidates for ADC, were subjected to additional analysis by ELISA with titration and cell viability assays. The titration ELISA used the same conditions as described above in the single spot ELISA, except that the 5-fold diluted supernatant was serially diluted 3-fold for a total of 8 wells. Conditions similar to those described above were used in the secondary antibody cytolysis assay with titration. In this case, the IgG concentrations in all supernatants were corrected for the IgG concentration of the clone with the lowest expression level (measured by anti-IgG coating titration ELISA). From this concentration, all supernatants were serially diluted 2-fold for a total of 6 dilution steps, and 15 μl of the supernatants were added to the plate. The analysis was carried out using Graphpad Prism software.

Example 2 Expression and Purification of Anti-ROR2 Antibodies

Expression Vectors: Antibody sequences defined above that bind to hROR2 were synthesized as DNA using GenScript (Piscataway, USA) and incorporated into an expression vector containing the appropriate restriction sites and the appropriate constant domain.

Expression and purification of kROR2 antibodies: Expression vectors were transfected into HEK293T cells using Lipofectamine® LTX reagent with PLUS™ reagent (Thermo Fisher Scientific, Reinach, Switzerland, 15388100); after incubation for 1 day (37° C., 5% CO ₂ , growth medium: Dulbecco's Modified Eagle's Medium (DMEM) with high glucose content (4.5 g/l), with L-glutamine with 10% (vol/ volume) of fetal bovine serum (FCS), 100 IU/ml penicillin-streptomycin-fungisone and 2 mM L-glutamine (all from Bioconcept, Allschwil, Switzerland)) cells were expanded under selection conditions (2 μg/ml puromycin (Sigma-Aldrich, Bux, St. Gallen, Switzerland, P8833-25 mg stock solution at 2 mg/ml)). Cells were separated and further expanded (37°C, 5% CO ₂ ); once confluent, tissue culture dishes were coated with 20 μg/ml poly-L-lysine (Sigma-Aldrich, P1524) for 2 hours at 37° C. and washed twice with PBS. The cells were then trypsinized and seeded in a 1:3 ratio onto poly-L-lysine-coated plates. Once confluent again, cells were washed with PBS followed by medium change to production medium (DMEM/F-12, Gibco/Thermo Fisher Scientific, 31330-03) supplemented with 1 μg/ml puromycin (Sigma, P8833), 100 IU/ml penicillin -streptomycin fungizone (Bioconcept), 161 μg/ml N-acetyl-L-cysteine (Sigma-Aldrich, A8199) and 10 μg/ml reduced L-glutathione (Sigma-Aldrich, G6529). The supernatant, which was collected biweekly and filtered (0.22 μm) to remove cells, was stored at 4°C until purification.

For purification, the filtered supernatant was loaded onto an equilibrated PBS HiTrap protein A column (GE Healthcare, Frankfurt am Main, Germany, 17-0405-01) or a JSR Amsphere™ protein A column (JSR Life Sciences, Leuven, Belgium, JWT203CE) and washed with PBS; elution was performed using 0.1 M glycine (pH 2.5) on an AEKTA pure system (GE Healthcare). Fractions were immediately neutralized with 1 M Tris-HCl buffer (pH 8.0) and analyzed for protein purity and integrity by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The protein containing fractions were mixed and buffer exchanged using Amicon filter units (Millipore, Schaffhausen, Switzerland, UFC 901008) to a dilution of 1:100 and then filter sterilized using a low retention filter (0.20 µm, Carl Roth, Karlsruhe, Germany, RA49.1).

The purity and integrity of the recombinant antibodies were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.

Example 3 Antibody Binding to hROR2 Using SPR

Surface plasmon resonance to measure the affinities of hROR2 antibodies for hROR2 was performed on a Biacore T200 instrument (GE Healthcare). Antibodies were captured using a CM5 Protein A Sensor Chip (GE Healthcare, 29127556) or Protein G was immobilized on a CM5 Sensor Chip. For affinity measurements, either purified anti-hROR2 antibodies or 293T supernatants containing anti-hROR2 antibodies were used. In all cases, anti-hROR2 antibodies were diluted to 1-3 µg/mL in 1x HBS-EP+ running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA (pH 7.4) and 0.05% (v/v) Tween 20) and subjected to capture for 30 seconds with a flow rate of 30 μl/min. hROR2-TwinStrep (from Example 1) was diluted in running buffer using 2-fold serial dilutions ranging from 40 nM to 2.5 nM.

Association and dissociation were measured at a flow rate of 30 μl/min. for 120 seconds and 200 seconds, respectively. The calculation of the rate constants of association (k _on ) and dissociation (k _off ) was based on the Langmuir 1:1 binding model. The equilibrium dissociation constant (K _d ) was calculated from k _off /k _on . SPR sensograms for the antibodies from Table 9 are shown in FIG. 4, and the values of K _d , k _on and k _off are presented in table 9.

Example 4 Mouse ROR2 and Cynomolgus ROR2 Expression

The expression of each of the mouse ROR2 (mROR2) and the cynomolgus monkey ROR2 (cROR2) was carried out according to the protocol similar to that provided for the expression of hROR2 in Example 1.

Example 5 mAb cross-reactivity with mouse ROR2 and cynomolgus RQR2

Antibody binding to hROR2, mouse ROR2 (mROR2), and cynomolgus ROR2 (cROR2) was assessed in an ELIS A-based assay. For this, ELIS A plates were coated with 50 μl of 2 μg/ml of the following molecules in sodium bicarbonate coating buffer ( 0.1 M Na ₂ CO ₃ , 0.1 M NaHCO ₃ , pH 9.6): (a) anti-human Fc antibody (Jackson Immunoresearch, 109-006-098), (b) hROR2-TwinStrep, (c) ROR2 cynomolgus-TwinStrep or (d) mouse ROR2-TwinStrep. After incubation for 1-2 days at 4°C, the plates were washed with PBS supplemented with 0.05% (v/v) Tween 20 and blocked with 150 μl of PBS supplemented with 0.05% (v/v) Tween 20 and 3 % bovine serum albumin (BSA). Thereafter, 3-fold or 4-fold dilutions of purified antibodies were added, starting at concentrations of 0.5 or 2 μg/ml, respectively, in PBS supplemented with 1% (w/v) bovine serum albumin (BSA) and 0.05% (v/v) Tween 20. After 1 hour incubation at 37° C., plates were washed with PBS supplemented with 0.05% (v/v) Tween 20 before adding HRP-conjugated anti-human IgG antibody (Jackson Immunoresearch, 109-036 -008). After additional washing of the plate, the plates were washed with 50 μl of Sigmafast OPD Tablet set (Sigma, P9187) and the reaction was stopped by adding 2 M sulfuric acid. The plates were read on an ELIS A (OD 490) plate reader.

In FIG. 5 and FIG. 11 shows the ELISA profiles for mAbs from Table 10. This table also summarizes the binding status for each mAb with ROR2 from each of the evaluated species.

Example 6 Conjugation of mAbs with Glycine Modified Toxins to Generate ADC Using SMAC™ Technology

Sortase A A recombinant and affinity purified enzyme sortase A from Staphylococcus aureus was produced in E. coli as disclosed in WO 2014140317 A1.

Creation of glycine-modified toxins. Pentaglycine-modified EDA-anthracycline derivative G5-PNU and triglycine-modified EDA-anthracycline derivative G3-PNU (FIGS. 6 (A) and (B), respectively) were manufactured by Concords, San Diego, USA. The identity and purity of the glycine-modified toxins was confirmed by mass spectrometry and HPLC. Each of the glycine-modified toxins exhibited >95% purity as determined by HPLC chromatography.

Sortase mediated antibody conjugation. The above toxins were conjugated to ROR2 antibodies and reference antibodies as indicated in Table 7 by incubating LPETG-labeled mAbs [5-10 µM] with glycine-modified toxin [100-200 µM] and 2.5-3 µM sortase A in the buffer provided. for conjugation for 3.5 hours at 25°C. The reaction was stopped by passing through a BioRad GraviTrap protein A column as shown in Table 11. The bound conjugate was eluted with 5 column volumes of elution buffer (0.1 M glycine, pH 2.5, 50 nM NaCl) with 1 volume fractions the columns were collected in tubes containing 25% v/v 1M Tris base to neutralize the acid. The protein-containing fractions were pooled and formulated with them in buffer to obtain the formulation of Table 11 using a ZebaSpin desalting column.

ADC analyses. DAR values were evaluated by reverse phase chromatography performed on a Polymer Labs PLRP 2.1 mm × 5 cm, 5 µm column with a flow rate of 1 ml/min./80°C with a 25-minute linear gradient from 0.05 to 0 .1% TFA/H ₂ O and 0.04 to 0.1% TFA/CH ₃ CN. Samples were first recovered by incubation with DTT at pH 8.0 and at 37° C. for 15 minutes. DAR values determined by reverse phase chromatography are summarized in Table 7 below.

From these assays, it can be concluded that SMAC™ conjugation proceeded with high efficiency, resulting in overall average DAR values ranging from about 3.5 to 4.0 for each of the hROR2 antibody-toxin combinations.

Example 7 Exposure of EMT-6 Cells to Stable Expression of hROR2 by Genetic Engineering Techniques

EMT-6 Cells Cultured in Complete DMEM (Dulbecco's Modified Eagle's Medium (DMEM) High Glucose (4.5 g/L) with L-Glutamine with 10% (v/v) Fetal Bovine Serum (FCS) , 100 IU/ml penicillin-streptomycin-fungisone and 2 mM L-glutamine (all from Bioconcept, Alschwil, Switzerland)) at 37° C. and 5% CO ₂ , centrifuged (6 minutes, 1200 rpm, 4 °C) and suspended in RPMI-1640 medium (5×10 ⁶ cells/ml). 400 µl of this cell suspension was then added to 400 µl of RPMI containing 10.2 µg of the pPB-PGK-Puro-hROR2-Thr transposable vector driving co-expression of the full-length ROR2 and the puromycin resistance gene and 3.6 µg of the pCDNA3.1_hy_mPB transposase vector. . A mixture of DNA/EMT-6 cells was transfected in electroporation cuvettes (0.4 cm lumen, 165-2088, BioRad, Cressier, Switzerland) and electroporated using a Biorad Gene Pulser II electroporator with a 300 V and 950 μF auxiliary capacitance module. The cells were then incubated for 5-10 minutes at room temperature. After incubation, the cells were centrifuged at 1200 rpm. for 6 minutes, washed once and then resuspended in complete DMEM before incubation at 37° C. in a humidified incubator with 5% CO ₂ . One day after electroporation, cell pools stably expressing hROR2 were selected for puromycin resistance using 3 μg/ml puromycin (Sigma-Aldrich, P8833-25 mg stock solution at 2 mg/ml).

ROR2 expression on selected EMT-6 ROR2 cells was confirmed by flow cytometry. Briefly, after trypsinization, 10b cells were centrifuged in FACS tubes; the resulting pellets were resuspended in buffer (PBS with 2% (v/v) FCS). Cells were then incubated with anti-ROR2 mAb Orb38364 (Biorbyt; 30 minutes, 4°C, final concentration 2 μg/ml) followed by centrifugation and washing. Cells were then resuspended as described above and incubated with PE-conjugated mouse anti-rabbit IgG (Abcam, ab99704) at a 1:250 dilution in the dark (30 minutes, 4°C) before washing.

Using FACS Aria II, cells were sorted as single cells into a 96-well flat-bottomed plate containing 200 μl of complete DMEM medium per well. This plate was incubated at 37°C and grown in 6 wells before screening. Cells were analyzed using the FACSCalibur instrument (BD Biosciences) and FlowJo analysis software (Tree Star, Ashland, OR, USA). In FIG. 7 shows FACS analysis of clone 14, which expresses ROR2 at a high level.

Example 8 In Vitro Cytotoxicity Assays of PNU Based ADCs on hROR2 Expressing EMT-6 Cancer Cells

The cytotoxicity of ADCs to ROR2 from Table 12 was studied using the genetically engineered EMT-6 cell line from Example 7.

For this, 1000 genetically engineered EMT6 cells (clone 14) per well were plated in 96-well plates (excluding the outer wells, which contained water) in 75 µl of DMEM supplemented with 10% v/v FCS, 100 IU/ml penicillin -streptomycin-fungisone and 2 mM L-glutamine, at a density of 1.3 x 105 cells per well, and grown at 37° C. in a humidified incubator with 5% CO ₂ . After 1 day incubation, each ADC was added to the respective wells at 25 μl of 3.5-fold serial dilutions in growth media (starting at an ADC concentration of 80 μg/ml, resulting in final ADC concentrations ranging from about 20 μg/ml to 0, 3 ng/ml). Each dilution was performed in duplicate. After 4 additional days, the plates were removed from the incubator and equilibrated at room temperature. After about 30 minutes, 50 μl was removed from each well, and then 50 μl of CellTiter-Glo® 2.0 luminescent solution (Promega, G9243) was added to each well. After shaking the plates at 750 rpm for 5 minutes followed by incubation for 20 minutes without shaking, luminescence was measured on a Spark 10 M plate reader with an integration time of 1 second per well. Curves of luminescence versus ADC concentration (ng/mL) were fitted using Graphpad Prism software. The measurement was repeated twice. IC ₅₀ values determined using the built-in IC ₅₀ determination function “log(inhibitor) vs. response -- Variable slope (four parameters))) in Prism Software are presented in Table 12.

In FIG. 8 is a dose-response curve of in vitro cytolysis assays on EMT-6 breast cancer cells stably expressing hROR2 using the PNU-based ADC of Table 12. Referring to Table 12 and FIG. 8 ADCs based on the novel anti-ROR2 antibodies of the present invention kill ROR2-expressing cells.

Example 9 In vitro cytotoxicity assays of PNU-based ADCs against hROR2-expressing EMT-6 breast cancer cells and against ROR2-negative cancer cells

Cytotoxicity of the ADCs to ROR2 of Table 13 was studied using the genetically engineered EMT-6 cell line of Example 7. The ROR2 low-expressing human cell line Karpas-299 was used as a control. ADC Ac10-G5-PNU was included as an isotype control.

For this, 1×10 ³ genetically engineered EMT6-ROR2 cells (clone 14) and 2.5×10 ³ Karpas-299 cells per well were plated in 96-well plates (excluding the outer wells, which contained water) at 75 µl of DMEM medium supplemented with 10% by volume FCS, 100 IU/ml penicillin-streptomycin-fungisone and 2 mM L-glutamine at a density of 1.3×10 ⁵ cells per well and grown at 37°C in a humidified incubator at 5 % CO ₂ . After 1 day incubation, each ADC was added to the respective wells in an amount of 25 μl of 3.5-fold serial dilutions in growth medium (starting at an ADC concentration of 80 μg/ml, resulting in final ADC concentrations ranging from 20 μg/ml to 0.89 ng/ml). Each dilution was performed in duplicate. After 4 additional days, the plates were removed from the incubator and equilibrated at room temperature. After about 30 minutes, 50 μl was removed from each well, and then 50 μl of CellTiter-Glo® 2.0 luminescent solution (Promega, G9243) was added to each well. After shaking the plates at 750 rpm. for 5 minutes followed by a 20 minute incubation without shaking, luminescence was measured on a Spark 10 M plate reader with an integration time of 1 second per well. Curves of luminescence versus ADC concentration (ng/mL) were fitted using Graphpad Prism software. IC ₅₀ values determined using the built-in IC ₅₀ determination function “log(inhibitor) vs. response -- Variable slope (four parameters))) in Prism Software are presented in Table 13.

Example 10 In Vitro Cytotoxicity Assays of PNU Based ADCs on hRQR2 Expressing EMT-6 Cancer Cells

The cytotoxicity of ADCs to ROR2 from Table 14 was studied using the genetically engineered EMT-6 cell line from Example 7.

For this, 1×10 ³ genetically engineered EMT6-ROR2 cells (clone 14) per well were plated on 96-well plates (except for the outer wells, which contained water) in 75 µl of DMEM supplemented with 10% by volume FCS, 100 IU/ml penicillin-streptomycin-fungisone and 2 mM L-glutamine, at a density of 1.3×10 ⁵ cells per well and grown at 37° C. in a humidified incubator with 5% CO ₂ . After 1 day incubation, each ADC was added to the respective wells in an amount of 25 μl of 3.5-fold serial dilutions in growth medium (starting at an ADC concentration of 80 μg/ml, resulting in final ADC concentrations ranging from 20 μg/ml to 0.89 ng/ml). Each dilution was performed in duplicate. After 4 additional days, the plates were removed from the incubator and equilibrated at room temperature. After about 30 minutes, 50 μl of CellTiter-Glo® 2.0 Luminescent Solution (Promega, G9243) was added to each well. After shaking the plates at 750 rpm. For 5 minutes followed by a 20 minute incubation without shaking, luminescence was measured on a Spark 10 M plate reader with an integration time of 1 second per well. Curves of luminescence versus ADC concentration (ng/mL) were fitted using Graphpad Prism software. IC ₅₀ values determined using the built-in IC ₅₀ determination function “log(inhibitor) vs. response -- Variable slope (four parameters))) in Prism Software are presented in Table 14.

Although the foregoing invention has been described in some detail for the purpose of illustration and example for the purpose of clarity of understanding, it will be obvious to a person skilled in the art, in view of the ideas of the present invention, that certain changes and modifications can be made therein without departing from the idea or scope. the appended claims.

All publications, databases, GenBank sequences, patents, and patent applications cited in this specification are incorporated herein by reference as if each were expressly and separately incorporated by reference.

SEQUENCES

This application also provides a sequence listing in electronic form according to WIPO ST 25. For the avoidance of doubt, if there are inconsistencies between the sequences in the following table and the electronic sequence listing, the sequences in this table will be considered correct.

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US Patent No. 4667014.

US Patent No. 4748034.

US Patent No. 5239660.

US Patent No. 3832253.

US Patent No. 3854480.

US Patent No. 4458066.

US Patent No. 8916159.

International application WO 2010/070263 A1.

International application WO 2014/013026 A1.

International application WO 2014/140317 A1.

International application WO 2016/102679 A1.

International application WO 2013/103637 A1.

International application WO 2016/142768 A1.

International application WO 2014/140317 A1.

--->

SEQUENCE LIST

<110> NBE-TERAPOITICS AG

<120> Human antibodies that bind to ROR2

<130>ND 40646

<160> 142

<170> BiSSAP 1.3.6

<210> 1

<211> 370

<212> Protein

<213> Artificial sequence

<220>

<223> human ROR2 extracellular domain

<400> 1

Glu Val Glu Val Leu Asp Pro Asn Asp Pro Leu Gly Pro Leu Asp Gly

1 5 10 15

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

20 25 30

Glu Pro Val Asn Asn Ile Thr Ile Val Gln Gly Gln Thr Ala Ile Leu

35 40 45

His Cys Lys Val Ala Gly Asn Pro Pro Asn Val Arg Trp Leu Lys

50 55 60

Asn Asp Ala Pro Val Val Gln Glu Pro Arg Arg Ile Ile Ile Arg Lys

65 70 75 80

Thr Glu Tyr Gly Ser Arg Leu Arg Ile Gln Asp Leu Asp Thr Thr Asp

85 90 95

Thr Gly Tyr Tyr Gln Cys Val Ala Thr Asn Gly Met Lys Thr Ile Thr

100 105 110

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

115 120 125

His Asn Phe Gln Asp Asp Tyr His Glu Asp Gly Phe Cys Gln Pro Tyr

130 135 140

Arg Gly Ile Ala Cys Ala Arg Phe Ile Gly Asn Arg Thr Ile Tyr Val

145 150 155 160

Asp Ser Leu Gln Met Gln Gly Glu Ile Glu Asn Arg Ile Thr Ala Ala

165 170 175

Phe Thr Met Ile Gly Thr Ser Thr His Leu Ser Asp Gln Cys Ser Gln

180 185 190

Phe Ala Ile Pro Ser Phe Cys His Phe Val Phe Pro Leu Cys Asp Ala

195 200 205

Arg Ser Arg Thr Pro Lys Pro Arg Glu Leu Cys Arg Asp Glu Cys Glu

210 215 220

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

225 230 235 240

Asn Pro Leu Ile Leu Met Arg Leu Gln Leu Pro Lys Cys Glu Ala Leu

245 250 255

Pro Met Pro Glu Ser Pro Asp Ala Ala Asn Cys Met Arg Ile Gly Ile

260 265 270

Pro Ala Glu Arg Leu Gly Arg Tyr His Gln Cys Tyr Asn Gly Ser Gly

275 280 285

Met Asp Tyr Arg Gly Thr Ala Ser Thr Thr Lys Ser Gly His Gln Cys

290 295 300

Gln Pro Trp Ala Leu Gln His Pro His Ser His His Leu Ser Ser Thr

305 310 315 320

Asp Phe Pro Glu Leu Gly Gly Gly His Ala Tyr Cys Arg Asn Pro Gly

325 330 335

Gly Gln Met Glu Gly Pro Trp Cys Phe Thr Gln Asn Lys Asn Val Arg

340 345 350

Met Glu Leu Cys Asp Val Pro Ser Cys Ser Pro Arg Asp Ser Ser Lys

355 360 365

Met Gly

370

<210> 2

<211> 450

<212> Protein

<213> Artificial sequence

<220>

<223>MK-3B12HC

<400> 2

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Gly

20 25 30

Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

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

50 55 60

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

65 70 75 80

Leu Lys Leu Thr Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Arg Ala Gly Trp Tyr Pro Phe Asp Cys Trp Gly Gln

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Gly Arg

450

<210> 3

<211> 213

<212> Protein

<213> Artificial sequence

<220>

<223>MK-3B12LC

<400> 3

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

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

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser 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

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Trp Thr

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Asn Arg Gly Glu Cys

210

<210> 4

<211> 448

<212> Protein

<213> Artificial sequence

<220>

<223>MK-7C3HC

<400> 4

Glu Val Gln Leu Leu Glu Thr Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Arg Ser His

20 25 30

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

35 40 45

Ala Leu Ile Trp Tyr Asp Gly Ser Lys Lys Tyr 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 Val Gly Ala Gly Leu Tyr Leu 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 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 Lys

435 440 445

<210> 5

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223>MK-7C3LC

<400> 5

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Ser Asn Trp

20 25 30

Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser 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

Asp Asp Phe Ala Ser Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser Tyr

85 90 95

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

100 105 110

Pro Ser Val Phe Ile Phe 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> 6

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> GK-1E5HC

<400> 6

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr

20 25 30

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

35 40 45

Ala Ile Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Thr Asp Ser Val

50 55 60

Gln 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 Pro Gly Ile Ala Met Thr Gly Leu Asp Tyr Trp Gly Gln Gly

100 105 110

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

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

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

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 7

<211> 213

<212> Protein

<213> Artificial sequence

<220>

<223> GK-1E5LC

<400> 7

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

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

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser 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

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Trp Thr

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Asn Arg Gly Glu Cys

210

<210> 8

<211> 453

<212> Protein

<213> Artificial sequence

<220>

<223> GK-5A1HC

<400> 8

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Asn Asp Gly Ser Asn Lys Tyr 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 Glu Gly Ser Gly Trp Tyr Asp Tyr Tyr Tyr Gly Met Asp Val

100 105 110

Trp Gly Glyn Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

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

275 280 285

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

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Leu Ser Pro Gly Lys

450

<210> 9

<211> 213

<212> Protein

<213> Artificial sequence

<220>

<223> GK-5A1 LC

<400> 9

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

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

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser 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

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Trp Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Asn Arg Gly Glu Cys

210

<210> 10

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> GK-2G8HC

<400> 10

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr

20 25 30

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

35 40 45

Ala Ile Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Thr 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 Pro Gly Val Ala Met Thr Gly Leu Asp Leu Trp Gly Gln Gly

100 105 110

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

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

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

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 11

<211> 213

<212> Protein

<213> Artificial sequence

<220>

<223> GK-2G8 LC

<400> 11

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

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

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser 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

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Trp Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Asn Arg Gly Glu Cys

210

<210> 12

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> GK-5E1HC

<400> 12

Gln Val Thr Leu Lys Glu Ser Gly Gly Asp Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Thr Tyr

20 25 30

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

35 40 45

Ala Leu Ile Trp Tyr Asp Gly Ser Lys Lys Tyr 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

Val Arg Val Arg Phe Gly Glu Leu Tyr Phe Gln His Trp Gly Gln Gly

100 105 110

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

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

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

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 13

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> GK-5E1LC

<400> 13

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

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

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser 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

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser Tyr

85 90 95

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

100 105 110

Pro Ser Val Phe Ile Phe 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> 14

<211> 448

<212> Protein

<213> Artificial sequence

<220>

<223> GK-6B10HC

<400> 14

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr

20 25 30

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

35 40 45

Ala Leu Ile Trp Tyr Asp Gly Ser Asn Lys Tyr 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 Val Ala Ala Ala Leu His Phe His 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 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 Lys

435 440 445

<210> 15

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> GK-6B10LC

<400> 15

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asp Asn Trp

20 25 30

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

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser 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

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser Tyr

85 90 95

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

100 105 110

Pro Ser Val Phe Ile Phe 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> 16

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> GK-5G12HC

<400> 16

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Thr Tyr

20 25 30

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

35 40 45

Ala Leu Ile Trp Tyr Asp Gly Ser Asn Lys Tyr 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

Ile Arg Val Lys Phe Gly Asp Leu Tyr Phe Gln His Trp Gly Gln Gly

100 105 110

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

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

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

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 17

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> GK-5G12LC

<400> 17

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser 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 Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asn Ser Ala Pro Tyr

85 90 95

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

100 105 110

Pro Ser Val Phe Ile Phe 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> 18

<211> 452

<212> Protein

<213> Artificial sequence

<220>

<223> GK-21D3HC

<400> 18

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

Ala Ile Ile Trp Tyr Asp Gly Ser Asn Lys Tyr 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 Asp Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Met Gly Ala Ile Asn Arg Gly Gly Gly Gly Phe Asp Tyr Trp

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Ser Pro Gly Lys

450

<210> 19

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> GK-21D3 LC

<400> 19

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser 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 Val Ser Thr Tyr Tyr Cys Gln Lys Tyr Asn Ser Ala Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe 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> 20

<211> 457

<212> Protein

<213> Artificial sequence

<220>

<223>MK-24C10HC

<400> 20

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

Ala Val Ile Trp Phe Asp Gly Thr Asn Lys His 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 Asp Lys Gly Glu Trp Phe Gly Glu Leu Arg Tyr Tyr Tyr Tyr

100 105 110

Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala

115 120 125

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

130 135 140

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

145 150 155 160

Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly

165 170 175

Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu

180 185 190

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

195 200 205

Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys

210 215 220

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

225 230 235 240

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

245 250 255

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

260 265 270

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

275 280 285

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

290 295 300

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

305 310 315 320

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

325 330 335

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

340 345 350

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

355 360 365

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

370 375 380

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

385 390 395 400

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

405 410 415

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

420 425 430

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

435 440 445

Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 21

<211> 219

<212> Protein

<213> Artificial sequence

<220>

<223>MK-24C10LC

<400> 21

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

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

180 185 190

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

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 22

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223>MK-24F9HC

<400> 22

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

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

35 40 45

Gly Asp Ile Asn His Ser Arg Thr Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

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

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Gly Glu Gln Trp Leu Val Pro Phe Asp Tyr Trp Asp Gln Gly

100 105 110

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

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

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

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 23

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223>MK-24F9LC

<400> 23

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser His Trp

20 25 30

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

35 40 45

Tyr Lys Ala Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Asn Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Asn Thr Tyr Ser Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe 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> 24

<211> 452

<212> Protein

<213> Artificial sequence

<220>

<223> GK-22G12HC

<400> 24

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Gln

20 25 30

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

35 40 45

Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Asn Tyr Val Asp Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Ile Ala Lys Asn Ser 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 Asp Gly Tyr Arg Asn Gly Trp His Ile Pro Glu Asp Tyr Trp

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Ser Pro Gly Lys

450

<210> 25

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> GK-22G12LC

<400> 25

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser 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 Val Ser Thr Tyr Tyr Cys Gln Lys His Asn Arg Ala Pro Trp

85 90 95

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

100 105 110

Pro Ser Val Phe Ile Phe 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> 26

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> MK-3B12HC CDR1

<400> 26

Gly Tyr Ser Ile Ser Ser Gly Tyr Tyr

fifteen

<210> 27

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223>MK-3B12HC CDR2

<400> 27

Ile Tyr Gln Ser Gly Ser Thr

fifteen

<210> 28

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> MK-3B12 HC CDR3

<400> 28

Cys Ala Arg Glu Asp Arg Ala Gly Trp Tyr Pro Phe Asp Cys Trp

1 5 10 15

<210> 29

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> MK-3B12 LC CDR1; GK-1E5 LC CDR1; GK-5A1 LC CDR1; GK-2G8 LC CDR1;

GK-5E1 LC CDR1; GK-1H2 LC CDR1; GK-2A9 LC CDR1; GK-5A6 LC CDR1

<400> 29

Gln Ser Ile Ser Ser Trp

fifteen

<210> 30

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> MK-3B12 LC CDR3; GK-1E5 LC CDR3; GK-2G8 LC CDR3; GK-1H2 LC CDR3;

GK-2A9 LC CDR3

<400> 30

Cys Gln Gln Tyr Asn Asn Tyr Trp Thr Phe

1 5 10

<210> 31

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> MK-7C3 HC CDR1

<400> 31

Gly Phe Thr Phe Arg Ser His Gly

fifteen

<210> 32

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> MK-7C3 HC CDR2; GK-1E5HC CDR2; GK-2G8HC CDR2; GK-5E1HC CDR2;

GK-1H2HC CDR2; GK-2A9HC CDR2

<400> 32

Ile Trp Tyr Asp Gly Ser Lys Lys

fifteen

<210> 33

<211> 13

<212> Protein

<213> Artificial sequence

<220>

<223> MK-7C3 HC CDR3

<400> 33

Cys Ala Arg Val Gly Ala Gly Leu Tyr Leu Asp Tyr Trp

1 5 10

<210> 34

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> MK-7C3 LC CDR1

<400> 34

Gln Thr Ile Ser Asn Trp

fifteen

<210> 35

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> MK-7C3 LC CDR3; GK-6B10 LC CDR3; GK-5A6 LC CDR3

<400> 35

Cys Gln Gln Tyr Asn Ser Tyr Ser Tyr Thr Phe

1 5 10

<210> 36

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> GK-1E5 HC CDR1; GK-2G8HC CDR1; GK-1H2HC CDR1; GK-2A9HC CDR1

<400> 36

Gly Phe Thr Phe Arg Ser Tyr Gly

fifteen

<210> 37

<211> 14

<212> Protein

<213> Artificial sequence

<220>

<223> GK-1E5 HC CDR3; GK-1H2HC CDR3

<400> 37

Cys Ala Arg Pro Gly Ile Ala Met Thr Gly Leu Asp Tyr Trp

1 5 10

<210> 38

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> GK-5A1 HC CDR1; GK-21D3HC CDR1; MK-24C10HC CDR1; GK-21F1HC

CDR1; MK-24C12HC CDR1

<400> 38

Gly Phe Thr Phe Ser Ser Tyr Gly

fifteen

<210> 39

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> GK-5A1 HC CDR2

<400> 39

Ile Trp Asn Asp Gly Ser Asn Lys

fifteen

<210> 40

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> GK-5A1 HC CDR3

<400> 40

Cys Ala Arg Glu Gly Ser Gly Trp Tyr Asp Tyr Tyr Tyr Gly Met Asp

1 5 10 15

Val Trp

<210> 41

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> GK-5A1 LC CDR3

<400> 41

Cys Gln Gln Tyr Asn Ser Tyr Trp Thr Phe

1 5 10

<210> 42

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> GK-5E1 LC CDR3

<400> 42

Cys Gln Gln Tyr Asn Ser Tyr Ser Tyr Ser Phe

1 5 10

<210> 43

<211> 14

<212> Protein

<213> Artificial sequence

<220>

<223> GK-2G8 HC CDR3; GK-5E1HC CDR1; GK-5G12HC CDR1; GK-2A9HC CDR3;

GK-5A6HC CDR1

<400> 43

Cys Ala Arg Pro Gly Val Ala Met Thr Gly Leu Asp Leu Trp

1 5 10

<210> 44

<211> 14

<212> Protein

<213> Artificial sequence

<220>

<223> GK-5E1 HC CDR3

<400> 44

Cys Val Arg Val Arg Phe Gly Glu Leu Tyr Phe Gln His Trp

1 5 10

<210> 45

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> GK-6B10 HC CDR1

<400> 45

Gly Phe Thr Phe Ser Arg Tyr Gly

fifteen

<210> 46

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> GK-6B10 HC CDR2; GK-5G12HC CDR2; GK-21D3HC CDR2; GK-5A6HC

CDR2; GK-21F1HC CDR2

<400> 46

Ile Trp Tyr Asp Gly Ser Asn Lys

fifteen

<210> 47

<211> 13

<212> Protein

<213> Artificial sequence

<220>

<223> GK-6B10 HC CDR3

<400> 47

Cys Ala Arg Val Ala Ala Ala Leu His Phe His Tyr Trp

1 5 10

<210> 48

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> >GK-6B10 LC CDR1

<400> 48

Gln Ser Ile Asp Asn Trp

fifteen

<210> 49

<211> 14

<212> Protein

<213> Artificial sequence

<220>

<223> >GK-5G12 HC CDR3; GK-5A6HC CDR3

<400> 49

Cys Ile Arg Val Lys Phe Gly Asp Leu Tyr Phe Gln His Trp

1 5 10

<210> 50

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> GK-5G12 LC CDR1; GK-21D3 LC CDR1; GK-22G12 LC CDR1; GK-21F1LC

CDR1; GK-21G5 LC CDR1; GK-21E6 LC CDR1; GK-22E12 LC CDR1

<400> 50

Gln Gly Ile Ser Asn Tyr

fifteen

<210> 51

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> GK-5G12 LC CDR3

<400> 51

Cys Gln Lys Tyr Asn Ser Ala Pro Tyr Thr Phe

1 5 10

<210> 52

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> GK-21D3 HC CDR3; GK-21F1HC CDR3

<400> 52

Cys Ala Arg Met Gly Ala Ile Asn Arg Gly Gly Gly Gly Phe Asp Tyr

1 5 10 15

trp

<210> 53

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> GK-21D3 LC CDR3; GK-21F1 LC CDR3

<400> 53

Cys Gln Lys Tyr Asn Ser Ala Pro Trp Thr Phe

1 5 10

<210> 54

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> MK-24C10 HC CDR2; MK-24C12HC CDR2

<400> 54

Ile Trp Phe Asp Gly Thr Asn Lys

fifteen

<210> 55

<211> 22

<212> Protein

<213> Artificial sequence

<220>

<223> MK-24C10 HC CDR3; MK-24C12HC CDR3

<400> 55

Cys Ala Arg Asp Lys Gly Glu Trp Phe Gly Glu Leu Arg Tyr Tyr Tyr

1 5 10 15

Tyr Gly Met Asp Val Trp

twenty

<210> 56

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> MK-24C10 LC CDR1; MK-24C12 LC CDR1

<400> 56

Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr

1 5 10

<210> 57

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> MK-24C10 LC CDR3; MK-24C12 LC CDR3

<400> 57

Cys Met Gln Ala Leu Gln Thr Pro Tyr Thr Phe

1 5 10

<210> 58

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> MK-24F9 HC CDR1; GK-21G5HC CDR1; GK-23A8HC CDR1

<400> 58

Gly Gly Ser Phe Ser Gly Tyr Tyr

fifteen

<210> 59

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> >MK-24F9 HC CDR2

<400> 59

Ile Asn His Ser Arg Thr Thr

fifteen

<210> 60

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> MK-24F9 HC CDR3

<400> 60

Cys Ala Arg Gly Gly Glu Gln Trp Leu Val Pro Phe Asp Tyr Trp

1 5 10 15

<210> 61

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> MK-24F9 LC CDR1; GK-23A8 LC CDR1

<400> 61

Gln Ser Ile Ser His Trp

fifteen

<210> 62

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> MK-24F9 LC CDR3; GK-23A8 LC CDR3

<400> 62

Cys Gln His Tyr Asn Thr Tyr Ser Arg Thr Phe

1 5 10

<210> 63

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> GK-22G12 HC CDR1; GK-21E6HC CDR1; GK-22E12HC CDR1

<400> 63

Gly Phe Thr Phe Ser Ser Gln Arg

fifteen

<210> 64

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> GK-22G12 HC CDR2; GK-21E6HC CDR2; GK-22E12HC CDR2

<400> 64

Ile Lys Gln Asp Gly Ser Glu Lys

fifteen

<210> 65

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> GK-22G12 HC CDR3; GK-21E6HC CDR3; GK-22E12HC CDR3

<400> 65

Cys Ala Arg Asp Gly Tyr Arg Asn Gly Trp His Ile Pro Glu Asp Tyr

1 5 10 15

trp

<210> 66

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> GK-22G12 LC CDR3; GK-21G5 LC CDR3; GK-21E6 LC CDR3; GK-22E12LC

CDR3

<400> 66

Cys Gln Lys His Asn Arg Ala Pro Trp Thr Phe

1 5 10

<210> 67

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> CDR1; X1=F or G, X2=T or S, X3=R or S, X4=S, T, R or G, X5=H, Y

or Q, X6=G, Y or R

<400> 67

Gly Xaa Xaa Phe Xaa Xaa Xaa Xaa

fifteen

<210> 68

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> CDR2; X7=W or K, X8=Y, N, F or Q, X9=S or T, X10=K, N or E

<400> 68

Ile Xaa Xaa Asp Gly Xaa Xaa Lys

fifteen

<210> 69

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> CDR1; X11=S, T or G, X12=S, N or H and X13=W or Y

<400> 69

Gln Xaa Ile Ser Xaa Xaa

fifteen

<210> 70

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> CDR3; X14=Q or M, X15=K, H or Q, X16=H, Y or A, X17=N or L,

X18=R, T, Q, S or N; X19=A, Y or T; X20=P, S or W, X21=W, R, Y or

missing, X22=S or T.

<400> 70

Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe

1 5 10

<210> 71

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> CDR1; X3=R or S, and X4=S, T

<400> 71

Gly Phe Thr Phe Xaa Xaa Tyr Gly

fifteen

<210> 72

<211> 4

<212> Protein

<213> Artificial sequence

<220>

<223> GK-5G12 LC CDR2; GK-21D3 LC CDR2; GK-22G12 LC CDR2; GK-21F1LC

CDR2; GK-21G5 LC CDR2; GK-21E6 LC CDR2; GK-22E12 LC CDR2

(sequence has only 3 AA (X is missing)

<400> 72

Ala Ala Ser Xaa

one

<210> 73

<211> 4

<212> Protein

<213> Artificial sequence

<220>

<223> MK-3B12 LC CDR2; MK-7C3 LC CDR2; GK-1E5 LC CDR2; GK-5A1 LC CDR2;

GK-2G8 LC CDR2; GK-5E1 LC CDR2; GK-6B10 LC CDR2; MK-24F9 LC CDR2;

GK-1H2 LC CDR2; GK-2A9 LC CDR2; GK-5A6 LC CDR2; GK-23A8 LC CDR2

(sequence has only 3 AA (X is missing)

<400> 73

Lys Ala Ser Xaa

one

<210> 74

<211> 4

<212> Protein

<213> Artificial sequence

<220>

<223> MK-24C10 LC CDR2, MK-24C12 LC CDR2 (sequence only has

3AA (X missing)

<400> 74

Leu Gly Ser Xaa

one

<210> 75

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> GK-1H2HC

<400> 75

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr

20 25 30

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

35 40 45

Ala Ile Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Thr 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 Pro Gly Ile Ala Met Thr Gly Leu Asp Tyr Trp Gly Gln Gly

100 105 110

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

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

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

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 76

<211> 213

<212> Protein

<213> Artificial sequence

<220>

<223> GK-1H2 LC

<400> 76

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

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

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser 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

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Trp Thr

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Asn Arg Gly Glu Cys

210

<210> 77

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> GK-2A9HC

<400> 77

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr

20 25 30

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

35 40 45

Ala Ile Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Thr 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 Pro Gly Val Ala Met Thr Gly Leu Asp Leu Trp Gly Gln Gly

100 105 110

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

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

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

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 78

<211> 213

<212> Protein

<213> Artificial sequence

<220>

<223> GK-2A9LC

<400> 78

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

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

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser 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

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Trp Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Asn Arg Gly Glu Cys

210

<210> 79

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> GK-5A6HC

<400> 79

Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Thr Tyr

20 25 30

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

35 40 45

Ala Leu Ile Trp Tyr Asp Gly Ser Asn Lys Tyr 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

Ile Arg Val Lys Phe Gly Asp Leu Tyr Phe Gln His Trp Gly Gln Gly

100 105 110

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

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

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

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 80

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> GK-5A6LC

<400> 80

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

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

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser 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

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser Tyr

85 90 95

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

100 105 110

Pro Ser Val Phe Ile Phe 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> 81

<211> 452

<212> Protein

<213> Artificial sequence

<220>

<223> GK-21F1HC

<400> 81

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

Ala Ile Ile Trp Tyr Asp Gly Ser Asn Lys Tyr 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 Asp Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Met Gly Ala Ile Asn Arg Gly Gly Gly Gly Phe Asp Tyr Trp

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Ser Pro Gly Lys

450

<210> 82

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> GK-21F1 LC

<400> 82

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser 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 Val Ser Thr Tyr Tyr Cys Gln Lys Tyr Asn Ser Ala Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe 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> 83

<211> 457

<212> Protein

<213> Artificial sequence

<220>

<223>MK-24C12HC

<400> 83

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

Ala Val Ile Trp Phe Asp Gly Thr Asn Lys His 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 Asp Lys Gly Glu Trp Phe Gly Glu Leu Arg Tyr Tyr Tyr Tyr

100 105 110

Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala

115 120 125

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

130 135 140

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

145 150 155 160

Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly

165 170 175

Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu

180 185 190

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

195 200 205

Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys

210 215 220

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

225 230 235 240

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

245 250 255

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

260 265 270

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

275 280 285

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

290 295 300

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

305 310 315 320

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

325 330 335

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

340 345 350

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

355 360 365

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

370 375 380

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

385 390 395 400

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

405 410 415

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

420 425 430

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

435 440 445

Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 84

<211> 219

<212> Protein

<213> Artificial sequence

<220>

<223>MK-24C12LC

<400> 84

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

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

180 185 190

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

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 85

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> GK-21G5HC

<400> 85

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

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

35 40 45

Gly Glu Ile Asn His Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

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

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Gly Asp Gln Trp Leu Val Pro Phe Asp Asn Trp Gly Gln Gly

100 105 110

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

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

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

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 86

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> GK-21G5LC

<400> 86

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser 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 Val Ser Thr Tyr Tyr Cys Gln Lys His Asn Arg Ala Pro Trp

85 90 95

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

100 105 110

Pro Ser Val Phe Ile Phe 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> 449

<212> Protein

<213> Artificial sequence

<220>

<223> GK-23A8HC

<400> 87

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

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

35 40 45

Gly Glu Ile Asn His Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

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

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Gly Asp Gln Trp Leu Val Pro Phe Asp Asn Trp Gly Gln Gly

100 105 110

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

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

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

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 88

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> GK-23A8LC

<400> 88

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser His Trp

20 25 30

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

35 40 45

Tyr Lys Ala Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Asn Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Asn Thr Tyr Ser Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe 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> 89

<211> 452

<212> Protein

<213> Artificial sequence

<220>

<223> GK-21E6HC

<400> 89

Arg Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Ser Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Gln

20 25 30

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

35 40 45

Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Asn Tyr Val Asp Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Ile Ala Lys Asn Ser 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 Asp Gly Tyr Arg Asn Gly Trp His Ile Pro Glu Asp Tyr Trp

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Ser Pro Gly Lys

450

<210> 90

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> GK-21E6LC

<400> 90

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser 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 Val Ser Thr Tyr Tyr Cys Gln Lys His Asn Arg Ala Pro Trp

85 90 95

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

100 105 110

Pro Ser Val Phe Ile Phe 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> 452

<212> Protein

<213> Artificial sequence

<220>

<223> GK-22E12HC

<400> 91

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Gln

20 25 30

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

35 40 45

Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Asn Tyr Val Asp Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Ile Ala Lys Asn Ser 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 Asp Gly Tyr Arg Asn Gly Trp His Ile Pro Glu Asp Tyr Trp

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Ser Pro Gly Lys

450

<210> 92

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> GK-22E12LC

<400> 92

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser 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 Val Ser Thr Tyr Tyr Cys Gln Lys His Asn Arg Ala Pro Trp

85 90 95

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

100 105 110

Pro Ser Val Phe Ile Phe 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> 93

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> GK-21G5 HC CDR2, GK-23A8 HC CDR2

<400> 93

Ile Asn His Ser Gly Ile Thr

fifteen

<210> 94

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> GK-5E1 HC CDR1; GK-5G12HC CDR1; GK-5A6HC CDR1

<400> 94

Gly Phe Thr Phe Arg Thr Tyr Gly

fifteen

<210> 95

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> GK-21G5 HC CDR3, GK-23A8 HC CDR3

<400> 95

Cys Ala Arg Gly Gly Asp Gln Trp Leu Val Pro Phe Asp Asn Trp

1 5 10 15

<210> 96

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Sequence for recognition by Staphylococcus aureus sortase A,

in which X is any amino acid

<400> 96

Leu Pro Xaa Ser Gly

fifteen

<210> 97

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> sequence for recognition by Staphylococcus aureus sortase A,

in which X is any amino acid

<400> 97

Leu Pro Xaa Ala Gly

fifteen

<210> 98

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> sequence for recognition by Staphylococcus aureus sortase A

or an engineered sortase A 4S-9 from Staphylococcus aureus in which X

is any amino acid

<400> 98

Leu Pro Xaa Thr Gly

fifteen

<210> 99

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> sequence for recognition by engineered sortase A 2A-9

from Staphylococcus aureus in which X is any amino acid

<400> 99

Leu Ala Xaa Thr Gly

fifteen

<210> 100

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> sequence for recognition by Staphylococcus aureus sortase A

or engineered sortase A 4S-9 from Staphylococcus aureus

<400> 100

Leu Ala Glu Thr Gly

fifteen

<210> 101

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> sequence for recognition by Streptococcus pyogenes sortase A,

in which X is any amino acid

<400> 101

Leu Pro Xaa Thr Ala

fifteen

<210> 102

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> sequence for recognition by Staphylococcus aureus sortase

<400> 102

Asn Pro Gln Thr Asn

fifteen

<210> 103

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> >huVH4B-3'Leader-FR1

<400> 103

caggtgcagc tgcaggagtc sg 22

<210> 104

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> huVH5B-3'-FR1 leader sequence

<400> 104

caggtacagc tgcagcagtc a 21

<210> 105

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> huVH6B-3'-FR1 leader sequence

<400> 105

caggtgcagc tacagcagtg gg 22

<210> 106

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> huVH10Ba-3'-leader sequence-FR1

<400> 106

gaggtgcagc tgktggagwc t 21

<210> 107

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> huVH10Bb-3'-FR1 leader sequence

<400> 107

gaggtgcagc tgktggagwc c 21

<210> 108

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> huVH12B-3'-FR1 leader sequence

<400> 108

caggtccagc tkgtrcagtc tgg 23

<210> 109

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> huVH14B-3'-FR1 leader sequence

<400> 109

cagrtcacct tgaaggagtc tg 22

<210> 110

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> huVH22B-3'-FR1 leader sequence

<400> 110

caggtgcagc tggtgsartc tgg 23

<210> 111

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> rat_IgG12abc_R

<400> 111

cagggtgact gagggcgtag 20

<210> 112

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> rat_IgM_R

<400> 112

gttgggaagg ttctgacacc 20

<210> 113

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> hu5'VK1-5_3'-leader sequence-FR1

<400> 113

gacatccaga tgacccagtc 20

<210> 114

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> hu5'VK1-9_3'-leader sequence-FR1

<400> 114

gacatccagt tgacccagtc t 21

<210> 115

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> hu5'VK1D-43_3'-leader sequence-FR1

<400> 115

gccatccgga tgacccagtc 20

<210> 116

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> hu5'VK2-24_3'-leader sequence-FR1

<400> 116

gatattgtga tgacccagac 20

<210> 117

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> hu5'VK2-28_3'-leader sequence-FR1

<400> 117

gatattgtga tgactcagtc 20

<210> 118

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> hu5'VK2-30_3'-leader sequence-FR1

<400> 118

gatgttgtga tgactcagtc 20

<210> 119

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> hu5'VK3-11_3'-leader sequence-FR1

<400> 119

gaaattgtgt tgacacagtc 20

<210> 120

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> hu5'VK3-15_3'-leader sequence-FR1

<400> 120

gaaatagtga tgacgcagtc 20

<210> 121

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> hu5'VK3-20_3'-leader sequence-FR1

<400> 121

gaaattgtgt tgacgcagtc t 21

<210> 122

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> hu5'VK4-1_3'-leader sequence-FR1

<400> 122

gacatcgtga tgacccagtc 20

<210> 123

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> rat_CK_R

<400> 123

cttgacactg atgtctctgg g 21

<210> 124

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> hu3'_JH1245_NheI

<400> 124

tgaggagacg gtgaccag 18

<210> 125

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> hu3'_JH3_NheI

<400> 125

tgaagagacg gtgaccattg 20

<210> 126

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> hu3'_JH6_NheI

<400> 126

tgaggagacg gtgaccgtg 19

<210> 127

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> huHSCJK2o-B_BsiWI

<400> 127

tttgatctcc agcttggtcc c 21

<210> 128

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> huHSCJK3o-B_BsiWI

<400> 128

tttgatatcc actttggtcc c 21

<210> 129

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> huHSCJK5o-B_BsiWI

<400> 129

tttaatctcc agtcgtgtcc c 21

<210> 130

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> huHSCJK14o-B_BsiWI

<400> 130

tttgatytcc accttggtcc c 21

<210> 131

<211> 212

<212> Protein

<213> Artificial sequence

<220>

<223> XBR1-402LC

<400> 131

Ser Tyr Glu Leu Thr Gln Leu Pro Ser Val Ser Val Ser Leu Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Glu Gly Asn Asn Ile Gly Ser Lys Ala Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Leu Ala Pro Gly Leu Leu Ile Tyr

35 40 45

Asp Asp Asp Glu Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asp Thr Ala Thr Leu Thr Ile Ser Gly Ala Gln Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ala Tyr Val

85 90 95

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

100 105 110

Pro Ser Val Thr Leu Phe Pro Ser Ser Glu Glu Glu Leu Gln Ala Asn

115 120 125

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

130 135 140

Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu

145 150 155 160

Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser

165 170 175

Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Lys Ser Tyr Ser

180 185 190

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

195 200 205

Thr Glu Cys Ser

210

<210> 132

<211> 447

<212> Protein

<213> Artificial sequence

<220>

<223> XBR1-402HC

<400> 132

Gln Glu Gln Gln Lys Glu Ser Gly Gly Gly Leu Phe Lys Pro Thr Asp

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Asp Ile Ser Ser Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Gly Ile Ser Gly Asn Ala Tyr Tyr Ala Ser Trp Ala Lys

50 55 60

Ser Arg Ser Thr Ile Thr Arg Asn Thr Asn Leu Asn Thr Val Thr Leu

65 70 75 80

Lys Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala

85 90 95

Arg Asp His Pro Thr Tyr Gly Met Asp Leu Trp Gly Pro Gly Thr Leu

100 105 110

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

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

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

195 200 205

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

210 215 220

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

275 280 285

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

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

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

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu 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 Val 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 Lys

435 440 445

<210> 133

<211> 447

<212> Protein

<213> Artificial sequence

<220>

<223> Ac10 LC

<400> 133

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

1 5 10 15

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

20 25 30

Tyr Ile Thr Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Tyr Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe

50 55 60

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

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Asn Tyr Gly Asn Tyr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Gln

100 105 110

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

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

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

195 200 205

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

210 215 220

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

275 280 285

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

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

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

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu 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 Val 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 Lys

435 440 445

<210> 134

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<223> Ac10 LC

<400> 134

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Phe Asp

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

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

180 185 190

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

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 135

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> linker derived from the sortase A recognition sequence

Staphylococcus aureus, in which X is any amino acid, and

n≥1 and ≤21

<400> 135

Leu Pro Xaa Thr Gly

fifteen

<210> 136

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> linker derived from the sortase A recognition sequence

Staphylococcus aureus, in which X is any amino acid, and

n≥1 and ≤21

<400> 136

Leu Pro Xaa Ala Gly

fifteen

<210> 137

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> linker derived from the sortase A recognition sequence

Staphylococcus aureus or engineered sortase A 4S-9 from Staphylococcus

aureus in which X is any amino acid and n≥1 and ≤21

<400> 137

Leu Pro Xaa Ser Gly

fifteen

<210> 138

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> linker derived from recognition sequence

constructed by sortase A 2A-9 from Staphylococcus aureus, in which X

is any amino acid, and n≥1 and ≤21

<400> 138

Leu Ala Xaa Thr Gly

fifteen

<210> 139

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> linker derived from the sortase A recognition sequence

Streptococcus pyogenes, in which X is any amino acid, and

n≥1 and ≤21

<400> 139

Leu Pro Xaa Thr Gly

fifteen

<210> 140

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> linker derived from sequence for sortase recognition

Staphylococcus aureus, in which n≥1 and ≤21

<400> 140

Asn Pro Gln Thr Gly

fifteen

<210> 141

<211> 370

<212> Protein

<213> Artificial sequence

<220>

<223> Cynomolgus monkey ROR2 extracellular domain (cROR2)

<400> 141

Glu Val Glu Val Pro Asp Pro Asn Asp Pro Leu Gly Pro Leu Asp Gly

1 5 10 15

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

20 25 30

Glu Pro Val Asn Asn Ile Thr Ile Val Gln Gly Gln Thr Ala Ile Leu

35 40 45

His Cys Lys Val Ala Gly Asn Pro Pro Asn Val Arg Trp Leu Lys

50 55 60

Asn Asp Ala Pro Val Val Gln Glu Pro Arg Arg Ile Ile Ile Arg Lys

65 70 75 80

Thr Glu Tyr Gly Ser Arg Leu Arg Ile Gln Asp Leu Asp Thr Thr Asp

85 90 95

Thr Gly Tyr Tyr Gln Cys Val Ala Thr Asn Gly Met Lys Thr Ile Thr

100 105 110

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

115 120 125

His Asn Phe Gln Asp Asp Tyr His Glu Asp Gly Phe Cys Gln Pro Tyr

130 135 140

Arg Gly Ile Ala Cys Ala Arg Phe Ile Gly Asn Arg Thr Ile Tyr Val

145 150 155 160

Asp Ser Leu Gln Met Gln Gly Glu Ile Glu Asn Arg Ile Thr Ala Ala

165 170 175

Phe Thr Met Ile Gly Thr Ser Thr His Leu Ser Asp Gln Cys Ser Gln

180 185 190

Phe Ala Ile Pro Ser Phe Cys His Phe Val Phe Pro Leu Cys Asp Ala

195 200 205

Arg Ser Arg Ala Pro Lys Pro Arg Glu Leu Cys Arg Asp Glu Cys Glu

210 215 220

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

225 230 235 240

Asn Pro Leu Ile Leu Met Arg Leu Gln Leu Pro Lys Cys Glu Ala Leu

245 250 255

Pro Met Pro Glu Ser Pro Asp Ala Ala Asn Cys Met Arg Ile Gly Ile

260 265 270

Pro Ala Glu Arg Leu Gly Arg Tyr His Gln Cys Tyr Asn Gly Ser Gly

275 280 285

Thr Asp Tyr Arg Gly Thr Ala Ser Thr Thr Lys Ser Gly His Gln Cys

290 295 300

Gln Pro Trp Ala Leu Gln His Pro His Ser His His Leu Ser Ser Thr

305 310 315 320

Asp Phe Pro Glu Leu Gly Gly Gly His Ala Tyr Cys Arg Asn Pro Gly

325 330 335

Gly Gln Met Glu Gly Pro Trp Cys Phe Thr Gln Asn Lys Asn Val Arg

340 345 350

Met Glu Leu Cys Asp Val Pro Ser Cys Ser Pro Arg Asp Ser Ser Lys

355 360 365

Met Gly

370

<210> 142

<211> 370

<212> Protein

<213> Artificial sequence

<220>

<223> mouse ROR2 extracellular domain (mROR2)

<400> 142

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

1 5 10 15

Pro Asp Ser Pro Leu Pro Thr Leu Lys Gly Tyr Phe Leu Asn Phe Leu

20 25 30

Glu Pro Val Asn Asn Ile Thr Ile Val Gln Gly Gln Thr Ala Ile Leu

35 40 45

His Cys Lys Val Ala Gly Asn Pro Pro Asn Val Arg Trp Leu Lys

50 55 60

Asn Asp Ala Pro Val Val Gln Glu Pro Arg Arg Val Ile Ile Arg Lys

65 70 75 80

Thr Glu Tyr Gly Ser Arg Leu Arg Ile Gln Asp Leu Asp Thr Thr Asp

85 90 95

Thr Gly Tyr Tyr Gln Cys Val Ala Thr Asn Gly Leu Lys Thr Ile Thr

100 105 110

Ala Thr Gly Val Leu Tyr Val Arg Leu Gly Pro Thr His Ser Pro Asn

115 120 125

His Asn Phe Gln Asp Asp Asp Gln Glu Asp Gly Phe Cys Gln Pro Tyr

130 135 140

Arg Gly Ile Ala Cys Ala Arg Phe Ile Gly Asn Arg Thr Ile Tyr Val

145 150 155 160

Asp Ser Leu Gln Met Gln Gly Glu Ile Glu Asn Arg Ile Thr Ala Ala

165 170 175

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

180 185 190

Phe Ala Ile Pro Ser Phe Cys His Phe Val Phe Pro Leu Cys Asp Ala

195 200 205

Arg Ser Arg Ala Pro Lys Pro Arg Glu Leu Cys Arg Asp Glu Cys Glu

210 215 220

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

225 230 235 240

Asn Pro Leu Ile Leu Met Arg Leu Gln Leu Pro Lys Cys Glu Ala Leu

245 250 255

Pro Met Pro Glu Ser Pro Asp Ala Ala Asn Cys Met Arg Ile Gly Ile

260 265 270

Pro Ala Glu Arg Leu Gly Arg Tyr His Gln Cys Tyr Asn Gly Ser Gly

275 280 285

Ala Asp Tyr Arg Gly Met Ala Ser Thr Thr Lys Ser Gly His Gln Cys

290 295 300

Gln Pro Trp Ala Leu Gln His Pro His Ser His Arg Leu Ser Ser Thr

305 310 315 320

Glu Phe Pro Glu Leu Gly Gly Gly His Ala Tyr Cys Arg Asn Pro Gly

325 330 335

Gly Gln Met Glu Gly Pro Trp Cys Phe Thr Gln Asn Lys Asn Val Arg

340 345 350

Val Glu Leu Cys Asp Val Pro Pro Cys Ser Pro Arg Asp Gly Ser Lys

355 360 365

Met Gly

370

<---

Claims (66)

1. A fully human antibody or fragment thereof that specifically binds to the extracellular domain of the tyrosine kinase-like orphan receptor 2 (ROR2), which are cross-reactive with (i) human ROR2 (hROR2) and (ii) cynomolgus monkey ROR2 (cROR2), which contain one of the following sets of CDRs: a) Heavy chain CDRs 1-3 as set forth in SEQ ID NOs: 38, 39 and 40, and light chain CDRs 1 and 3 as set forth in SEQ ID NOs: 29 and 41, with light chain CDR 2 having the sequence KAS , b) heavy chain CDRs 1-3 as set forth in SEQ ID NOs: 26-28 and light chain CDRs 1-3 as set forth in SEQ ID NOs: 29, 73 and 30, c) heavy chain CDRs 1-3 as set forth in SEQ ID NOs: 94, 32 and 44 and light chain CDRs 1 and 3 as set forth in SEQ ID NOs: 29 and 42, with light chain CDR 2 having the sequence KAS , d) heavy chain CDRs 1-3 as set forth in SEQ ID NOS: 38, 46 and 52 and light chain CDRs 1 and 3 as set out in SEQ ID NOS: 50 and 53, with light chain CDR 2 having the sequence AAS , e) heavy chain CDRs 1-3 as set forth in SEQ ID NOs: 38, 54 and 55 and light chain CDRs 1 and 3 as set forth in SEQ ID NOs: 56 and 57, with light chain CDR 2 having the sequence LGS , f) heavy chain CDRs 1-3 as set forth in SEQ ID NOs: 58-60 and light chain CDRs 1 and 3 as set forth in SEQ ID NOs: 61 and 62, with light chain CDR 2 having the sequence KAS, g) heavy chain CDRs 1-3 as set forth in SEQ ID NOs: 63-65, with light chain CDRs 1 and 3 as set forth in SEQ ID NOs: 50 and 66, with light chain CDRs 2 having the sequence AAS, and the CDRs are contained in a suitable protein scaffold so that they are capable of binding to hROR2 as well as cynomolgus ROR2 with sufficient affinity. 2. An antibody or fragment according to claim 1, which contains one of the following pairs of sequences: a) the sequence of the heavy chain variable region of the GK-5A1 antibody shown in SEQ ID NO: 8 and the light chain variable region sequence of the antibody GK-5A1 shown in SEQ ID NO: 9, b) the heavy chain variable region sequence of the MK-3B12 antibody as set forth in SEQ ID NO: 2 and the light chain variable region sequence of the MK-3B12 antibody as set forth in SEQ ID NO: 3, c) the heavy chain variable region sequence of the GK-5E1 antibody as set forth in SEQ ID NO: 12 and the light chain variable region sequence of the GK-5E1 antibody as set forth in SEQ ID NO: 13, d) the sequence of the heavy chain variable region of the GK-21D3 antibody shown in SEQ ID NO: 18 and the light chain variable region sequence of the antibody GK-5E1 shown in SEQ ID NO: 19, e) the MK-24C10 antibody heavy chain variable region sequence as set forth in SEQ ID NO: 20 and the MK-24C10 antibody light chain variable region sequence as set forth in SEQ ID NO: 21, f) the sequence of the heavy chain variable region of the antibody MK-24F9 as set forth in SEQ ID NO: 22 and the sequence of the light chain variable region of the antibody MK-24F9 as set forth in SEQ ID NO: 23, g) the heavy chain variable region sequence of the GK-22G12 antibody as set forth in SEQ ID NO: 24 and the light chain variable region sequence of the GK-22G12 antibody as set forth in SEQ ID NO: 25. 3. An antibody-drug conjugate (ADC) for the treatment of neoplastic disease, having the general formula A-(L)n-(T)m, wherein A is an antibody or fragment thereof according to any one of paragraphs. 12, L is a linker, T is the payload as a cytotoxic or cytostatic agent and in which n and m are integers from >1 to <10. 4. An antibody-effector molecule conjugate (AEC) for the treatment of neoplastic disease, having the general formula A-(L)n-(T)m, wherein A is an antibody or fragment thereof according to any one of paragraphs. 12, L is a linker, T is a label and in which n and m are integers from >1 to <10. 5. The conjugate according to any one of paragraphs. 3 and 4, wherein the linker contains or consists of at least one selected from the group consisting of oligopeptide linker, a maleimide linker, optionally containing cleavable spacers that can be cleaved by changes in pH, redox potential and/or specific intracellular enzymes. 6. The conjugate according to any one of paragraphs. 3-5, wherein the linker has at least one of the following amino acid sequences: -LPXTGn-, -LPXAGn-, -LPXSGn-, -LAXTGn-, -LPXTGn-, -LPXTAn- or -NPQTGn-, wherein Gn is an oligo- or polyglycine, wherein n is an integer from ≥1 to ≤21, An is an oligo- or polyalanine, while n is an integer from ≥1 to ≤21, and X is any possible amino acid sequence. 7. The conjugate according to any one of paragraphs. 3-6, wherein the linker is conjugated to the C-terminus of at least one subdomain of the antibody or fragment thereof. 8. The conjugate according to any one of paragraphs. 3-7, and before conjugation the antibody or fragment thereof carries a sortase recognition motif fused or conjugated to the C-terminus of at least one of its subdomains, and the toxin or label contains a glycine segment ≥1 to ≤21 glycine residues in length, preferably ≥2 to ≤5 glycine residues in length. 9. Conjugate antibody-drug according to any one of paragraphs. 3-8, wherein the toxin is at least one selected from the group consisting of: maytansinoids, auristatins, anthracyclines, preferably anthracyclines derived from PNU-159682, calicheamicins, tubulizins, duocarmycins, radioactive isotopes, liposomes containing a payload in the form of toxoid, protein toxins, taxanes, pseudodimers of indilino-benzodiazepine and/or pyrrolobenzodiazepines, or is their derivative. 10. The conjugate according to any one of paragraphs. 3-9, which is produced by sortase-mediated conjugation of (i) an antibody bearing one or more sortase recognition motifs, and (ii) one or more toxins or labels bearing an oligoglycine tag. 11. The method of obtaining a conjugate according to any one of paragraphs. 3-10, the method comprising the following steps: a) providing an antibody or fragment thereof according to any one of paragraphs. 1, 2, wherein the antibody carries one or more sortase recognition motifs, b) providing one or more toxins or labels bearing an oligoglycine tag, and c) conjugation of the antibody and the toxin or label by sortase-mediated conjugation. 12. The use of an antibody-drug conjugate according to any one of paragraphs. 3 or 5-10 for the manufacture of a medicament for the treatment of neoplastic disease. 13. Use according to claim 12, wherein the neoplastic disease is at least one selected from the group consisting of renal cell carcinoma, osteosarcomas, kidney cancer. 14. Pharmaceutical composition for the treatment of neoplastic disease, containing an antibody-drug conjugate according to any one of paragraphs. 3 or 5-10 together with one or more pharmaceutically acceptable ingredients. 15. A method of killing or inhibiting the growth of a cell expressing ROR2 in a patient, the method comprising administering to the cell a pharmaceutically effective amount or dose of (i) an antibody-drug conjugate according to any one of paragraphs. 3 or 5-10 or (ii) the pharmaceutical composition of claim 14, wherein the cell expressing ROR2 is a cancer cell. 16. Method for determining whether whether the intended patient suffers from a neoplastic disease or an immune disease and/or whether the intended patient is at risk of developing a neoplastic disease or an immune disease, said method comprising treating a sample taken from the subject with an antibody-effector molecule conjugate according to any one of paragraphs. 4-8 or 10. 17. The method of claim 16, wherein the neoplastic disease or immune disease is suitable for treatment with an anti-ROR2 antibody or fragment or anti-ROR2 antibody drug conjugate.

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