ROR1 antibody immunoconjugates

This application is a continuation of U.S. patent application Ser. No. 16/016,238, filed on Jun. 22, 2018, which claims priority from U.S. Patent Applications 62/524,382, 62/524,386, and 62/524,388, all of which were filed on Jun. 23, 2017. The disclosures of these priority applications are incorporated by reference herein in their entirety.

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 5, 2018, is named 024651_C1002_SL.txt and is 56,687 bytes in size.

Cancer is the second leading cause of human death next to coronary artery disease. Receptor tyrosine kinases (RTKs) play a key role in oncogenic transformation, growth and metastases. RTKs regulate cell differentiation, proliferation, migration, angiogenesis, and survival. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an evolutionarily-conserved type I membrane protein that belongs to the ROR subfamily and has extracellular domains that contain immunoglobulin (Ig)-like, Frizzled, and Kringle domains. ROR1-deficient mice display a variety of phenotypic defects within the skeletal and urogenital systems, as well as postnatal growth retardation. ROR1 is expressed during embryogenesis and by a variety of different cancers, but not by normal post-partum tissues, and can be considered an onco-embryonic surface antigen. Functional data suggest that ROR1 may function in non-canonical WNT-signaling to promote the survival of malignant cells.

ROR1 expression and activation appears to be correlated with features of tumor aggressiveness in models of chronic lymphocytic leukemia (CLL), breast cancer, lung cancer, gastric cancer, and melanoma (Li et al., PLoS One 5(7):e11859 (2010); Gentile et al., Cancer Res. 71(8):3132-41 (2011); Zhang et al., PLoS One 7(3):e31127 (2012); Yamaguchi et al., Cancer Cell. 21(3):348-61 (2012); Daneshmanesh et al., Leukemia 26(6):1348-55 (2012); Daneshmanesh et al., Leuk Lymphoma 54(4):843-50 (2013); O'Connell et al., Cancer Discov. 3(12):1378-93 (2013); Hojjat-Farsangi et al., PLoS One 8(4):e61167 (2013); Hojjat-Farsangi et al., PLoS One 8(10):e78339 (2013); Ida et al., Cancer Sci. 107(2):155-61 (2016); and Janovska et al., Clin Cancer Res. 22(2):459-69 (2016)). Elevated levels of ROR1 expression in patients and cell lines are associated with genes involved in epithelial-mesenchymal transition (EMT) (Cui et al., Cancer Res. 73(12):3649-60 (2013)). In patients with CLL, high levels of ROR1 expression are associated with shorter treatment-free survival and overall survival (OS) (Cui et al., Blood 128(25):2931-2940 (2016)). Similarly, in patients with ovarian cancer, high ROR1 expression is associated with poor clinical outcomes (Zhang et al., Sci Rep. 4:5811 (2014)).

In view of the role of ROR1 in cancer, there is a need for new and improved therapies that target ROR1-positive cancer cells.

Provided herein is an immunoconjugate having the formula of Ab-((L)m-(D))n, wherein: Ab is an antibody or an antigen-binding fragment thereof that specifically binds to human receptor tyrosine kinase like orphan receptor 1 (ROR1); L is a linker, and m is 0 or 1; D is a cytotoxic drug moiety; and n is an integer from 1 to 10.

The cytotoxic drug moiety may be selected from the group consisting of, for example, an anti-tubulin agent, a DNA alkylating agent, a DNA cross-linking agent, a DNA intercalating agent, and an RNA polymerase II inhibitor. In some embodiments, the cytotoxic drug moiety is selected from the group consisting of monomethyl auristatin E (MMAE), azonafide, α-amanitin, duocarmycin TM, pyrrolobenzodiazepine (PBD), PNU-159682, and pharmaceutically acceptable salts, esters, and analogs thereof.

The linker in the immunoconjugate may comprise a cleavable moiety. It may be cleaved inside a target cell. Alternatively, the linker is not cleavable. The linker can be branched or unbranched. In some embodiments, the linker comprises one or more moieties selected from valine-citrulline (VC), valine-alanine (VA), para-aminobenzyloxycarbonyl (PAB), polyethylene glycol (PEG), diaminopropionic acid (DPR), Phe-C, C-Gly, Calkyl, dimethylethylamine (DMEA), and ethylene diamine (EDA). In certain embodiments, the linker is covalently bonded to the antibody or antigen-binding fragment at a succinimide, a carbonyl, or a cyclooctene, or a triazole group of the linker.

In certain embodiments, the antibody or fragment in the immunoconjugate is covalently bonded to the linker by reaction with a moiety selected from the group consisting of 6-maleimidocaproyl (MC)-VC-PAB; 6-MC-C; 6-MC-PEG4-VC-PAB-DMEA; 6-MC-PEG4-VA; 6-MC-DPR-VC-PAB; 6-MC-Phe-C-VC-PAB; 6-MC-Phe-C-VC-PAB-DMEA; 6-MC-C-Gly-EDA; dibenzylcyclooctyne (DBCO)-(PEG2-VC-PAB); DBCO-PEG4-VC-PAB-DMEA; and N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate-VC-PAB. As used herein, VC represents a valine-citrulline dipeptide; VA represents a valine-alanine dipeptide; PEG represents polyethylene glycol; PAB represents para-amino-benzyloxycarbonyl; DMEA represents dimethylethylamine; Phe represents a benzyl group; and EDA represents ethylene diamine.

Provided herein also is an immunoconjugate having the formula of Ab-((L)m-(D))n, wherein: Ab is an antibody or an antigen-binding fragment thereof that specifically binds to human receptor tyrosine kinase like orphan receptor 1 (ROR1); L is a cleavable linker, and m is 0 or 1; D is an auristatin (e.g., MMAE); and n is an integer from 1 to 10.

In an immunoconjugate of the present disclosure, the linker may comprise, for example, a heterocycle or carbonyl covalently bonded to the antibody or antigen-binding fragment, a spacer group covalently bonded to the heterocycle or carbonyl, and an ester, thioester, amide, carbonate, thiocarbonate or carbamate covalently bonded to the cytotoxic drug moiety. In some embodiments, the spacer group comprises an amino acid, a polyamino acid, or an amino benzyl group, or a combination thereof. In some embodiments, the linker in an immunoconjugate of the present disclosure forms a covalent bond with a cysteine or lysine residue on the antibody or fragment.

The Ab (antibody or fragment thereof) component of an immunoconjugate of the present disclosure may bind to the same ROR1 epitope as an antibody comprising the heavy chain and light chain amino acid sequences of SEQ ID NOs: 3 and 4, respectively. The antibody or fragment may comprise the heavy chain complementarity-determining region (CDR) 1-3 (HCDR1-3) in SEQ ID NO: 3 and the light chain CDR1-3 (LCDR1-3) in SEQ ID NO: 4. In some embodiments, the antibody or fragment comprises the amino acid sequences of SEQ ID NOs: 7-9, and the light chain of the antibody comprises the amino acid sequences of SEQ ID NOs: 10-12. The antibody or fragment may be humanized. The antibody or fragment may have one or more of the following properties: a) facilitates ROR1 internalization in a human cell; b) binds to human ROR1 with a Kof less than 100 nM (e.g., less than 50, 40, 30, 20, or 10 nM); and c) inhibits growth of ROR1human cancer cells in vitro with an ECof 500 nM or less (e.g., 400 nM or less, 300 nM or less, 200 nM or less, or 100 nM or less).

In some embodiments, the heavy chain variable domain (V) and light chain variable domain (V) of the antibody in the immunoconjugate comprise the amino acid sequences of: a) SEQ ID NOs: 5 and 6, respectively; b) SEQ ID NOs: 5 and 50, respectively; c) SEQ ID NOs: 48 and 6, respectively; or d) SEQ ID NOs: 48 and 50, respectively. The antibody may comprise a human IgGconstant region and optionally also a human κ light chain constant region. In further embodiments, the heavy chain and light chain of the antibody comprise the amino acid sequences of: a) SEQ ID NOs: 3 and 4, respectively; b) SEQ ID NOs: 3 and 49, respectively; c) SEQ ID NOs: 47 and 4, respectively; or d) SEQ ID NOs: 47 and 49, respectively.

In some embodiments, the Ab component of the immunoconjugate is an Fab, F(ab), or scFv, e.g., an Fab, F(ab), or scFv.

Specific embodiments of the present disclosure include an immunoconjugate comprising an antibody conjugated to a cytotoxic drug moiety, wherein the Vand Vof the antibody comprise the amino acid sequences of SEQ ID NOs: 5 and 6, respectively. Examples of such an immunoconjugate are shown in Tables 2 and 3 below, and include Antibody-Drug Conjugates (ADC)-A, E, H, I, J, K, L, M, N, O, P, Q, and R. In further embodiments, the heavy chain and light chain of the antibody comprise the amino acid sequences of SEQ ID NOs: 3 and 4, respectively.

In the immunoconjugate of the present disclosure, the number of the drug moiety to per antibody or fragment, or the ratio of the cytotoxic drug moiety to the antibody or fragment (DAR), may be 1 to 10, for example, 1 to 7, 1 to 6, 1 to 5, 2 to 7, 2 to 6, or 2 to 5.

Also provided herein are pharmaceutical compositions comprising an immunoconjugate of the present disclosure and a pharmaceutically acceptable excipient. The pharmaceutical compositions may further comprise an additional therapeutic agent selected from the group consisting of a Bruton's tyrosine kinase (BTK) inhibitor, a B-cell lymphoma 2 (Bcl-2) inhibitor, a mammalian target ofrapamycinerapamycin(mTOR) inhibitor, and a phosphoinositide 3-kinase (PI3K) inhibitor. For example, the additional therapeutic agent is selected from ibrutinib, acalabrutinib, venetoclax, everolimus, sapanisertib, and idelalisib.

Also provided herein is a therapy or method for treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an immunoconjugate of the present invention. The cancer may be homogenous or heterogeneous for ROR1 expression and may be, for example, a leukemia, a lymphoma, or a solid tumor. In some embodiments, the cancer is chronic lymphocytic leukemia (CLL), T-cell leukemia (TCL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma, multiple myeloma (MM), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), or a non-Hodgkin lymphoma (NHL) that has undergone Richter's transformation. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), hepatocellular carcinoma, pancreatic cancer, osteosarcoma, head and neck cancer, ovarian cancer, breast cancer, or triple negative breast cancer (TNBC).

The therapy or treatment method of the present disclosure may further comprise administering to the patient an additional anti-cancer therapeutic agent, which may be, for example, a Bruton's tyrosine kinase (BTK) inhibitor, a B-cell lymphoma 2 (Bcl-2) inhibitor, a mammalian target ofrapamycinerapamycin(mTOR) inhibitor, and a phosphoinositide 3-kinase (PI3K) inhibitor. In some embodiments, the additional therapeutic agent is selected from ibrutinib, acalabrutinib, venetoclax, everolimus, sapanisertib, and idelalisib.

In certain embodiments of the present therapy or treatment method, the cancer is CLL, MCL, or an NHL that has undergone Richter's transformation.

Provided herein also are immunoconjugates and pharmaceutical compositions as described herein for use in treating cancer in the therapy or treatment methods described herein. For example, provided herein is an immunoconjugate having the formula of Ab-((L)m-(D))n for use in treating cancer in a patient in need thereof, wherein: Ab is an antibody or an antigen-binding fragment thereof that specifically binds to human receptor tyrosine kinase like orphan receptor 1 (ROR1); L is a linker, and m is 0 or 1; D is a cytotoxic drug moiety; and n is an integer from 1 to 10. Exemplary embodiments of the immunoconjugate and the treatment are described above and will be further described below.

Provided herein also are the use of an immunoconjugate herein for the manufacture of a medicament for use in treating cancer in a patient in need thereof. For example, provided herein is the use of is an immunoconjugate having the formula of Ab-((L)m-(D))n for the manufacture of a medicament in treating cancer in a patient in need thereof, wherein: Ab is an antibody or an antigen-binding fragment thereof that specifically binds to human receptor tyrosine kinase like orphan receptor 1 (ROR1); L is a linker, and m is 0 or 1; D is a cytotoxic drug moiety; and n is an integer from 1 to 10. Exemplary embodiments of the immunoconjugate and the treatment are described above and will be further described below.

The present disclosure also provides a method of making an immunoconjugate, comprising: providing an antibody or an antigen-binding fragment thereof that specifically binds to human receptor tyrosine kinase like orphan receptor 1 (ROR1); conjugating to the antibody a cytotoxic drug moiety selected from the group consisting of an anti-tubulin agent, a DNA alkylating agent, a DNA cross-linking agent, a DNA intercalating agent, and an RNA polymerase II inhibitor; wherein the heavy chain of the antibody comprises the amino acid sequences of SEQ ID NOs: 7-9, and the light chain of the antibody comprises the amino acid sequences of SEQ ID NOs: 10-12. Exemplary embodiments of the immunoconjugate are described above and will be further described below.

Provided herein also are articles of manufactures, such as kits, comprising an immunoconjugate of the present disclosure.

The present invention provides immunoconjugates of the formula Ab-((L)-(D)), wherein Ab is an antibody or an antigen-binding fragment thereof that specifically binds to the ROR1 protein; L is a linker; D is a drug moiety that has therapeutic activity in cancer; m is 0 or 1; and n is an integer from 1 to 10. In the formula, the dash “-” denotes a covalent or non-covalent bond. The antibody or fragment includes, but is not limited to, an antibody or antibody fragment that competes with antibody D10 or Ab1 for binding to human ROR1, or binds to the same epitope as D10 or Ab1. The drug moiety includes, but is not limited to, another antibody or an antigen-binding fragment thereof, a polypeptide, a small molecule compound, a nucleic acid molecule such as a small interfering RNA molecule or an antisense molecule. The immunoconjugates of the present invention may be used to treat a variety of cancers such as ROR1-positive cancers.

1. Immunoconiugates

An “antibody-drug conjugate,” or “ADC,” or “immunoconjugate” refers to an antibody molecule, or an antigen-binding fragment thereof, that is covalently or non-covalently bonded, with or without a linker, to one or more biologically active molecule(s). The present immunoconjugates comprise antibodies or fragments thereof that are specific for human ROR1 and can thus serve as excellent targeting moieties for delivering the conjugated payloads to ROR1-positive cells. In some embodiments, a ROR1 immunoconjugate provided herein has an equilibrium dissociation constant (K) of about 1 μM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, or 0.001 nM or less (e.g., 10M or less, from 10M to 10M, or from 10M to 10M) for human ROR1. Kcan be measured by any suitable assay, such as surface plasmon resonance assays (e.g., using a BIACORE®-2000 or a BIACOREg-3000). In certain embodiments, the Kof an immunoconjugate of the invention is less than the Kfor the D10 antibody. In certain embodiments, the Kof an immunoconjugate of the invention for human ROR1 is less than about 50, 40, 30, 20, or 10 nM (e.g., 40 nM). In some embodiments, a ROR1 immunoconjugate provided herein inhibits growth of ROR1human cancer cells in vitro with an ECof about 500, 400, 350, 300, or 250 nM or less (e.g., 300 nM or less). As used herein, an antibody is said to bind specifically to an antigen when it binds to the antigen with a Kof 100 nM or less, such as less than 10 nM or less (e.g., 1-5 nM), as determined by, e.g., surface plasmon resonance or Bio-Layer Interferometry.

In certain embodiments, the immunoconjugate provided herein is internalized by a ROR1-positive cell primarily through the lysosome/endosome pathway. In particular embodiments, the internalization is independent of the ROR1 expression level on the cell surface.

Embodiments of the antibody or fragment thereof, the linker, and the drug moiety used in the immunoconjugates are described in further detail below.

1.1. Types and Structures of Antibodies

The term “antibody” is used herein in the broadest sense and includes polyclonal and monoclonal antibodies, such as intact antibodies and functional (antigen-binding) fragments thereof. The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multi-specific (e.g., bispecific) antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, and tandem tri-scFv. Unless otherwise indicated, the term encompasses intact or full-length antibodies, including antibodies of any class or subclass (e.g., IgG and sub-classes thereof such as IgG, IgG, IgG, and IgG; IgM; IgE; IgA; and IgD), as well as antibody fragments.

An antibody may include a heavy chain (or a polypeptide sequence derived therefrom) and a light chain (or a polypeptide sequence derived therefrom). The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in the antibody's binding to an antigen. The variable domains of the heavy chain and light chain (Vand V, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions and three complementarity-determining regions. A single Vor Vdomain may sometimes be sufficient to confer all or a majority of the antigen-binding specificity of an antibody. Furthermore, antibodies that bind a particular antigen may be isolated by using a Vor Vdomain from an antibody that binds the antigen to screen a library of complementary Vor Vdomains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The terms “complementarity-determining region” and “CDR,” which are synonymous with “hypervariable region” or “HVR,” refer to subregions within the antibody variable domains, which confer the antibody's specificity and/or affinity for its antigen. In general, there are three CDRs in each heavy chain variable domain (HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variable domain (LCDR1, LCDR2, and LCDR3). “Framework regions” (“FRs”) refer to the non-CDR portions of the variable domains. In general, there are four FRs in each full-length heavy chain variable domain and four FRs in each full-length light chain variable domain. The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of several well-known schemes, including those described by Kabat et al., 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991) (“Kabat” numbering scheme); Al-Lazikani et al., IMB 273, 927-948 (1997) (“Chothia” numbering scheme); MacCallum et al., J Mol. Biol. 262:732-745 (1996) (“contact” numbering scheme); Lefranc et al., Dev Comp Immunol. 27(1):55-77 (2003) (“IMGT” numbering scheme); and Honegger and Pluckthun, J Mot Biol, 309(3):657-70 (2001) (“Aho” numbering scheme).

The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on sequence alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a.” The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. The contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. Unless indicated otherwise, the CDRs of the antibodies referred to herein may be identified according to any of the Kabat, Chothia, IMGT, and contact methods.

An antigen-binding fragment of a full-length antibody may be used in making an immunoconjugate of the present invention. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab′, Fab′-SH, F(ab′); recombinant IgG (rIgG) fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv or sFv); single domain antibodies (e.g., sdAb, sdFv, nanobodies); and multi-specific antibodies formed from antibody fragments. In certain embodiments, the fragments are single-chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFvs.

1.2 Exemplary ROR1 Antibodies

An immunoconjugate of the invention comprises an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, e.g., human ROR1. The antibody or fragment binds to an extracellular portion of the ROR1 protein such as an epitope in one or more of the immunoglobulin (Ig)-like, Frizzled, and Kringle domains of the ROR1 protein. In certain embodiments, the ROR1-binding antibody or fragment binds to an amino acid sequence of ROR1 shown in SEQ ID NO: 1 or 2 (not including the terminal cysteine, which is added for convenience of conjugation) and can be internalized by a ROR1cell; examples of such an antibody are murine antibodies D10 and 99961. See U.S. Pat. Nos. 9,217,040 and 9,758,591, the disclosures of which are incorporated by reference herein in their entirety. In certain embodiments, the antibody or fragment competes with D10 or 99961 for binding to human ROR1. Amino acid sequences of exemplary anti-ROR1 antibodies used in the immunoconjugates of the invention are shown in Table 1 below, where Ab1-Ab4 are humanized variants of antibody 99961.

In some embodiments, the antibody or antibody fragment in the immunoconjugate specifically binds human ROR1, and its heavy and light chains respectively comprise:

In certain embodiments, the immunoconjugate of the invention comprises an anti-ROR1 antibody, or an antigen-binding fragment thereof, wherein the antibody comprises:

In certain embodiments, the Vand Vof the antibody respectively comprise the amino acid sequences of:

In certain embodiments, the HC and LC of the antibody respectively comprise the amino acid sequences of:

In certain embodiments, the immunoconjugate of the invention comprises an antibody or fragment thereof derived from a murine antibody with the Vand Vamino acid sequences of (i) SEQ ID NOs: 25 and 26, respectively; (ii) SEQ ID NOs: 35 and 36, respectively; or (iii) SEQ ID NOs: 45 and 46, respectively. Antibodies derived from these sequences may be, e.g., antibodies that have been humanized or joined to a human Fc region (e.g., chimeric). For example, the antibody or an antigen-binding fragment in the immunoconjugate comprises:

Exemplary coding sequences for the aforementioned antibodies are shown in Table 12 below. For example, the antibody in the immunoconjugate may comprise:

In certain embodiments, the immunoconjugate of the invention comprises an antigen-binding fragment of an anti-ROR1 antibody, wherein the antigen-binding fragment comprises the sequence of any one of SEQ ID NOs: 64-68. In certain embodiments, the antigen-binding fragment comprises the Vand Vamino acid sequences of:

Percent (%) sequence identity with respect to a reference polypeptide sequence refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known; for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR). For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

In some embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. A variant typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants can be naturally occurring or can be synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the invention and evaluating one or more biological activities of the polypeptide as described herein and/or using any of a number of known techniques. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.

As used herein, the term “substantially identical” refers to two or more sequences having a percentage of sequential units (e.g., amino acid residues) which are the same when compared and aligned for maximum correspondence over a comparison window, or a designated region as measured using comparison algorithms. By way of example, two or more sequences may be “substantially identical” if the sequential units are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, or about 99% identical over a specified region. Such percentages describe the “percent identity” between two sequences.

1.4 Making and Modification of ROR1 Antibodies