WO2025029920A1

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Publication number: WO2025029920A1
Application number: PCT/US2024/040391
Authority: WO
Inventors: Thomas J. Kipps; II George F. Widhopf; James B. Breitmeyer; Gunnar Kaufmann
Original assignee: University of California Berkeley; University of California San Diego UCSD; Oncternal Therapeutics Inc
Current assignee: University of California Berkeley; University of California San Diego UCSD; Oncternal Therapeutics Inc
Priority date: 2023-08-01
Filing date: 2024-07-31
Publication date: 2025-02-06
Anticipated expiration: 2026-02-01

Abstract

Provided herein are an ROR1 binding antibody or an ROR1 antigen binding fragment thereof and methods of manufacture and use, wherein the ROR1 binding antibody or the ROR1 antigen binding fragment thereof comprises an alteration to increase antibody effector function. The alteration to increase antibody effector function may increase antibody dependent cell cytotoxicity (ADCC), and the alteration to increase antibody effector function comprises glycoengineering.

Description

GLYCOENGINEERED FORMS OF ROR1 ANTIBODIES AND METHODS OF USE

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63/517,088 filed on August 1, 2023, and U.S. Provisional Application Serial No. 63/607,456 filed on December 7, 2023. Each of which is incorporated by reference herein in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically and is hereby incorporated by reference in its entirety. Said copy, created on July 5, 2024 is named 51956-728.601_SL.xml and is 10800 bytes in size.

BACKGROUND

[0003] Cancer is a leading cause of death worldwide. In the United States alone, cancer causes the death of well over a half-million people annually, with some 1.4 million new cases diagnosed per year. Receptor tyrosine kinases (RTKs) play critical roles in cell differentiation, proliferation, migration, angiogenesis, and survival. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a type I membrane protein that belongs to the ROR subfamily and has extracellular domains that contain immunoglobulin (Ig)-like, Frizzled, and Kringle domains. Recent advances have shed light on the role ROR1 may play in non- canonical WNT-signaling to promote the survival of malignant cells. Studies have also shown that non-canonical WNT signaling plays a major role in basal -like and other subtypes of cancer metastasis. The ability to develop antibodies targeting ROR1 across various cell types which hare suitable for clinical application is an important step in the development of therapies targeting ROR1.

SUMMARY

[0004] The pharmaceutical use of antibodies has increased over the past decades. However, many antibodies fail clinically notwithstanding their ability to bind a target antigen because they cannot bind all cell types (e.g., cancers) which express the target antigen with therapeutic efficacy. The challenge in developing therapeutic antibodies is that, generally, the amount of antibody that achieving antibody dependent cellular cytotoxicity (ADCC) efficacious treatment of diseases, for example, cancers, based on the amount of antibody which can be administered to a subject is limited by the activity of specific antibodies against cell types expressing corresponding antigens (e.g., R0R1) which are recognized by the antibody. For example, even if an antibody can bind a particular antigen (e.g., R0R1), the antibody may not achieve antibody dependent cellular cytotoxicity (ADCC) against all target cells expressing the antigen in order to be an effective treatment. Thus, one problem to be solved is how to develop improved antibodies binding particular targets which achieve mAb- dose-dependent ADCC against various target cell types expressing target at therapeutically achievable dosages.

[0005] R0R1 binding antibodies fail to achieve mAb-dose-dependent ADCC against R0R1 expressing cancers to a level which been shown to be efficacious in the treatment of against R0R1 expressing cancers. For example, zilovertamab has shown minimal ADCC acting alone, or only shows modest levels of ADCC in combination with other chemotherapeutic agents, and it is suggested that the epitope of ROR1 at the IgG like domain of human ROR1 may be a low density epitope expressed on leukemic cells that is a poor target for ADCC activity for an anti-RORl antibody acting alone. Afucosylation of zilovertamab significantly increases the ADCC activity of the modified antibody alone to a level which is comparable to that other approved mAb therapies which target high density epitopes in leukemic cancers. Surprisingly and unexpectedly, afucosylation of zilovertamab significantly increases the ADCC activity of the modified antibody, even when acting alone, to a level which is comparable to that rituximab, an approved antibody which targets a high- density epitope at CD20 expressed on leukemic cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0008] FIG. 1 shows activation of Jurkat-Lucia NFAT-CD16 cells by cells treated with anti-RORl or anti-CD20 mAb.

[0009] FIG. 2 shows activation of Jurkat-Lucia NFAT-CD16 cells by cells treated with anti-RORl or anti-CD20 mAb.

[0010] FIG. 3 shows activation of Jurkat-Lucia NFAT-CD16 cells by cells treated with anti-RORl or anti-CD20 mAb.

[0011] FIG. 4 shows antibody dependent cellular cytotoxicity (ADCC) by NK92-CD16v cells for target cells treated with anti-RORl or anti-CD20 mAb.

[0012] FIG. 5 shows ADCC by NK92-CD16v cells for ROR1+ CLL cells treated with anti-RORl or anti-CD20 mAb.

[0013] FIG. 6 shows ADCC by NK92 or NK92-CD16v cells for ROR1+ CLL cells treated with anti-RORl or anti-CD20 mAb.

[0014] FIG. 7 shows ADCC by NK92-CD16v cells for 17p deleted and p53 mutated ROR1+ CLL cells treated with anti- ROR1 or anti-CD20 mAb.

[0015] FIG. 8 shows ADCC by PBMC for ROR1+ CLL cells treated with anti-RORl or anti-CD20 mAb.

[0016] FIG. 9 shows ADCC by PBMC for ROR1+ CLL cells treated with anti-RORl or anti-CD20 mAb that can be blocked by addition of anti-human CD 16 mAb.

DETAILED DESCRIPTION

[0017] The pharmaceutical use of antibodies has increased over the past decades. However, many antibodies fail clinically notwithstanding their ability to bind a target antigen because they cannot bind all cell types (e.g., cancers) which express the target antigen with therapeutic efficacy. The challenge in developing therapeutic antibodies is that, generally, the amount of antibody that achieving antibody dependent cellular cytotoxicity (ADCC) efficacious treatment of diseases, for example, cancers, based on the amount of antibody which can be administer to a subject is limited by the activity of specific antibodies against cell types expressing corresponding antigens (e.g., ROR1) which are recognized by the antibody. For example, even if an antibody can bind a particular antigen (e.g., ROR1), the antibody may not achieve antibody dependent cellular cytotoxicity (ADCC) against all target cells expressing the antigen in order to be an effective treatment. Thus, one problem to be solved is how to develop improved antibodies binding particular targets which achieve mAb- dose-dependent ADCC against various target cell types expressing target at therapeutically achievable dosages.

[0018] ROR1 binding antibodies fail to achieve mAb-dose-dependent ADCC against ROR1 expressing cancers to a level which is efficacious in the treatment of against ROR1 expressing cancers. For example, zilovertamab shows minimal ADCC acting alone, or only shown modest levels of ADCC in combination with other chemotherapeutic agents, and it is suggested that the epitope of ROR1 at the IgG like domain of human ROR1 may be a low- density epitope expressed on leukemic cells that is a poor target for ADCC activity for an anti-RORl antibody acting alone. Afucosylation of zilovertamab significantly increases the ADCC activity of the modified antibody alone to a level which is comparable to that other approved mAb therapies which target high density epitopes in leukemic cancers. Surprisingly and unexpectedly, afucosylation of zilovertamab significantly increases the ADCC activity of the modified antibody, even when acting alone, to a level which is comparable to that rituximab, an approved antibody which targets a high-density epitope at CD20 expressed on leukemic cells.

Anti-RORl Antibodies

[0019] Provided herein are stable antibody formulations, and antibodies and antibody fragments that bind ROR1 in stable formulation. For example, provided herein are anti-RORl antibodies that, in certain instances, inhibit cancer cell growth and metastasis. In some embodiments, the anti-RORl antibody compositions are useful in methods for inhibiting metastasis using anti-RORl antibodies or antibody fragments thereof. In some embodiments, the antibody binds to the Ig-like domain, which is contiguous with the CRD domain of human ROR1 (hRORl). In certain embodiments, the anti-RORl antibodies bind to an epitope mapping to amino acids 42-160 of hRORl. In some embodiments, the anti-RORl antibodies bind to an epitope mapping to amino acids 130-160 of hRORl.

[0020] Aspects disclosed herein provide an ROR1 binding antibody or an ROR1 antigen binding fragment thereof, wherein the ROR1 binding antibody or the ROR1 antigen binding fragment thereof comprises an alteration to increase antibody effector function. In some embodiments, the alteration to increase antibody effector function increases antibody dependent cell cytotoxicity (ADCC). In some embodiments, the alteration to increase antibody effector function comprises one or more amino acid alterations to the Fc region of the antibody compared to a wild type antibody Fc region. In some embodiments, the one or more amino acid alterations comprise S239D, I332E, A330L, S239D/I332E;

[0021] Provided herein are stable pharmaceutical formulations of anti-RORl antibodies or RORl-binding antibody fragments thereof comprising:

(a) a heavy chain complementarity determining region 1 (H-CDR1) comprising an amino acid sequence set forth in SEQ ID NO: 1;

(b) a heavy chain complementarity determining region 2 (H-CDR2) comprising an amino acid sequence set forth in SEQ ID NO: 2;

(c) a heavy chain complementarity determining region 3 (H-CDR3) comprising an amino acid sequence set forth in SEQ ID NO: 3;

(d) a light chain complementarity determining region 1 (L-CDR1) comprising an amino acid sequence set forth in SEQ ID NO: 4;

(e) a light chain complementarity determining region 2 (L-CDR2) comprising an amino acid sequence SGS; and/or

(f) a light chain complementarity determining region 3 (L-CDR3) comprising an amino acid sequence set forth in SEQ ID NO: 6.

[0022] Further provided are stable pharmaceutical formulations of anti-RORl antibodies or RORl-binding antibody fragments thereof comprising:

(a) a heavy chain complementarity determining region 1 (H-CDR1);

(b) a heavy chain complementarity determining region 2 (H-CDR2);

(c) a heavy chain complementarity determining region 3 (H-CDR3); (d) a light chain complementarity determining region 1 (L-CDR1);

(e) a light chain complementarity determining region 2 (L-CDR2); and/or

(f) a light chain complementarity determining region 3 (L-CDR3); wherein the H-CDR1 , the H-CDR2, and the H-CDR3 are derived from SEQ ID NO: 7, and the L-CDR1, the L-CDR2, and the L-CDR3 are derived from SEQ ID NO: 8; and wherein the H-CDR1, the H-CDR2, the H-CDR3, L-CDR1, the L-CDR2, and the L-CDR3 are defined using Chothia, Kabat, IMGT, Contact, or AbM methodologies.

[0023] In some embodiments, the anti-RORl antibodies or ROR1 -binding antibody fragments thereof comprise: a heavy chain variable domain comprising an amino acid sequence having 70% or greater sequence identity to SEQ ID NO: 7; and a light chain variable domain comprising an amino acid sequence having 70% or greater sequence identity to SEQ ID NO: 8.

[0024] In some embodiments, the heavy chain variable domain comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO: 7. In some embodiments, the heavy chain variable domain comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 7. In some embodiments, the heavy chain variable domain comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO:

7. In some embodiments, the heavy chain variable domain comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO: 7. In some embodiments, the heavy chain variable domain comprises an amino acid sequence having 98% or greater sequence identity to SEQ ID NO: 7. In some embodiments, the heavy chain variable domain comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 7.

[0025] In some embodiments, the light chain variable domain comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO: 8. In some embodiments, the light chain variable domain comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 8. In some embodiments, the light chain variable domain comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO:

8. In some embodiments, the light chain variable domain comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO: 8. In some embodiments, the light chain variable domain comprises an amino acid sequence having 98% or greater sequence identity to SEQ ID NO: 8. In some embodiments, the light chain variable domain comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 8.

[0026] In some embodiments, the anti-RORl antibodies or ROR1 -binding antibody fragments thereof comprise: a heavy chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO: 7; and a light chain variable domain comprising an amino acid as set forth in SEQ ID NO: 8.

[0028] In some embodiments, the anti-RORl antibodies comprise: a heavy chain domain comprising an amino acid sequence having 70% or greater sequence identity to SEQ ID NO: 9; and a light chain domain comprising an amino acid sequence having 70% or greater sequence identity to SEQ ID NO: 10.

[0029] In some embodiments, the heavy chain domain comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO: 9. In some embodiments, the heavy chain domain comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 9. In some embodiments, the heavy chain domain comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 9. In some embodiments, the heavy chain domain comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO: 9. In some embodiments, the heavy chain domain comprises an

- I l amino acid sequence having 98% or greater sequence identity to SEQ ID NO: 9. In some embodiments, the heavy chain domain comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 9.

[0030] In some embodiments, the light chain domain comprises an amino acid sequence having 80% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the light chain domain comprises an amino acid sequence having 85% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the light chain domain comprises an amino acid sequence having 90% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the light chain domain comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the light chain domain comprises an amino acid sequence having 98% or greater sequence identity to SEQ ID NO: 10. In some embodiments, the light chain domain comprises an amino acid sequence having 99% or greater sequence identity to SEQ ID NO: 10.

[0031] In some embodiments, the anti-RORl antibodies comprise: a heavy chain domain comprising an amino acid sequence as set forth in SEQ ID NO: 9; and a light chain domain comprising an amino acid as set forth in SEQ ID NO: 10. In some embodiments, the anti-RORl antibody is zilovertamab (previously known as cirmtuzumab).

Altered Anti-RORl Antibodies; Glycoengineered Anti-RORl Antibodies

[0033] In some cases, the alteration or modification is a mutation. In some cases, the antibody further comprises one or more mutations in a framework region, e.g., in the CHI domain, CH2 domain, CH3 domain, hinge region, or a combination thereof. In some instances, the one or more mutations are to stabilize the antibody and/or to increase half-life. In some instances, the one or more mutations are to modulate Fc receptor interactions, to increase ADCC or complement-dependent cytotoxicity (CDC). In other instances, the one or more mutations are to reduce or eliminate Fc effector functions such as FcyR-binding, ADCC, or CDC. In additional instances, the one or more mutations are to modulate glycosylation, e.g., fucosylation. In some cases, the one or more mutations enhance stability, increase half-life, decrease glycosylation, and/or modulate Fc receptor interactions, e.g., to increase or decrease ADCC and/or CDC.

[0034] In some cases, the antibody comprises an IgGl framework. In some embodiments, the constant region of the antibody is modified at one or more amino acid positions to alter Fc receptor interaction. Exemplary residues that modulate or alter Fc receptor interaction include, but are not limited to, G236, S239, T250, M252, S254, T256, K326, A330, 1332, E333A, M428, H433, or N434 (Kabat numbering; EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest). In some instances, the mutation comprises G236A, S239D, T250Q, M252Y, S254T, T256E, K326W, A330L, I332E, E333A, E333S, M428L, H433K, or N434F.

[0035] In some embodiments, the modification at one or more amino acid positions in the IgGl constant region to alter Fc receptor interaction leads to increased half-life. In some instances, the modification at one or more amino acid positions comprise T250, M252, S254, T256, M428, H433, N434, or a combination thereof; e.g., comprising T250Q/M428L or M252Y/S254T/T256E and H433K/N434F. In some embodiments, the IgGl constant region is modified at amino acid N297 (Kabat numbering) in which residue N297 is deglycosylated. [0036] In some embodiments, the antibody comprises an IgG4 framework. In some instances, one or more amino acid positions in the IgG4 framework are modified to alter Fc receptor interaction, e.g., to increase ADCC and/or CDC. For example, mutations to increase ADCC comprises, in some embodiments, S239D, I332E, and A330L (amino acid numbering is according to the EU index in Kabat et al), such as described in U.S. Patent No. 8,093,359. In some cases, one or more amino acid positions in the IgG4 framework are modified to stabilize the antibody and/or to increase half-life. In some instances, one or more amino acid positions in the IgG4 framework are modified to modulate glycosylation. In some cases, the constant region is modified at a hinge region to prevent or reduce strand exchange. In some instances, the amino acid that is modified is S228 (e.g., S228P).

[0037] In some embodiments, the antibodies are altered to increase or decrease their glycosylation (e.g., by altering the amino acid sequence such that one or more glycosylation sites are created or removed). A carbohydrate attached to an Fc region of an antibody may be altered. Native antibodies from mammalian cells typically comprise a branched, biantennary oligosaccharide attached by an N-linkage to Asn297 of the CH2 domain of the Fc region (See e.g., Wright et al. TIBTECH 15 :26-32 (1997)). The oligosaccharide can be various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, sialic acid, fucose attached to a GlcNAc in the stem of the biantennar oligosaccharide structure. Modifications of the oligosaccharide in an antibody can be made, for example, to create antibody variants with certain improved properties. Antibody glycosylation variants can have improved ADCC and/or CDC function. In some embodiments, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn297 (See e.g., WO 08/077546). Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues; See e.g., Edelman et al. Proc Natl Acad Sci U S A. 1969 May; 63(1): 78-85).

However, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants can have improved ADCC function (See e.g., Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); and Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)). Cell lines, e.g., knockout cell lines and methods of their use can be used to produce afucosylated antibodies, e.g., Lecl3 CHO cells deficient in protein fucosylation and alpha-1, 6-fucosyltransferase gene (FUT8) knockout CHO cells (See e.g., Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006)). Other antibody glycosylation variants are also included (See e.g., U.S. Pat. No. 6,602,684). [0038] Zilovertamab is a first in class antibody for targeting an epitope of R0R1, which is a potential target for treatment R0R1 expressing cancers. However, previously studies have shown minimal ADCC with zilovertamab alone, or only shown modest levels of ADCC in combination with other chemotherapeutic agents (e.g., BCL2 inhibitors) (Rassenti L. et al., Proc. Natl. Acad. Sci. USA, 114: 10733, 2017). Previous studies of zilovertamab have failed to achieve levels of ADCC which were comparable to that of other approved antibodies for other targets on leukemic cells (e.g., CD20), and considering that zilovertamab fails to recognize other ROR1 epitopes recognized by competing anti-RORl antibodies, the epitope of ROR1 at the IgG like domain of human ROR1 may be a low density epitope expressed on leukemic cells such that it is a poor target for ADCC activity acting alone. Surprisingly and unexpectedly, afucosylation of zilovertamab significantly increases the ADCC activity of the modified antibody alone to a level which is comparable to that rituximab, an approved antibody which targets a high density epitope at CD20 expressed on leukemic cells. When considering the previous studies in which zilovertamab has failed to achieve appreciable ADCC when acting alone, and the low epitope density of the epitope of ROR1 at the IgG like domain of human ROR1 (e.g., including glutamic acid at position 138), one of skill in the art would not have expected that afucosylation of zilovertamab would significantly increase the ADCC of the modified antibody to a level which is comparable to that of a high density epitope therapeutic, such an anti-CD20 antibody like rituximab.

[0039] In certain embodiments described herein are a method of making a glycoengineered antibody comprising culturing a cell expressing a nucleic acid encoding an ROR1 binding antibody or an ROR1 antibody binding fragment thereof of the current disclosure under conditions that result in reduced fucosylation or afucosylation, and isolating and/or purifying the antibody from the cell or the cell culture media.

Pharmaceutical Formulations

[0040] Provided herein are pharmaceutical compositions comprising the anti-RORl antibodies described herein. A composition or pharmaceutical formulation or pharmaceutical composition generally encompasses and/or refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Pharmaceutical formulations are generally sterile (e.g., aseptic or free from all living microorganisms and their spores). The pharmaceutical compositions described and provided herein generally comprise, in some embodiments, the anti-RORl antibody or RORl-binding antibody fragment compounded with one or more of: a buffer, a chelator, a surfactant, and a stabilizer (e.g., a tonicity or osmolarity adjusting agent). [0041] In certain embodiments the anti-RORl antibody or RORl-binding antibody fragments of the current disclosure are included in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, and diluents. Pharmaceutically acceptable excipients, carriers and diluents can be included to increase shelf-life, stability, or the administrability of the antibody. Such compounds include salts, pH buffers, detergents, anti-coagulants, and preservatives. In certain embodiments, the antibodies of the current disclosure are administered suspended in a sterile solution. In certain embodiments, the solution comprises about 0.9% NaCl. In certain embodiments, the solution comprises about 5.0% dextrose. In certain embodiments, the solution further comprises one or more of: buffers, for example, acetate, citrate, histidine, succinate, phosphate, bicarbonate and hydroxymethylaminomethane (Tris); surfactants, for example, polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and pol oxamer 188; polyol/disaccharide/polysaccharides, for example, glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; amino acids, for example, glycine or arginine; antioxidants, for example, ascorbic acid, methionine; or chelating agents, for example, EDTA or EGTA.

[0042] In certain embodiments, the antibodies of the current disclosure can be shipp ed/stored lyophilized and reconstituted before administration. In certain embodiments, lyophilized antibody formulations comprise a bulking agent such as, mannitol, sorbitol, sucrose, trehalose, dextran 40, or combinations thereof. The lyophilized formulation can be contained in a vial comprised of glass or other suitable non-reactive material. The antibodies when formulated, whether reconstituted or not, can be buffered at a certain pH, generally less than 7.0. In certain embodiments, the pH can be between 4.5 and 7.0, 4.5 and 6.5, 4.5 and 6.0, 4.5 and 5.5, 4.5 and 5.0, or 5.0 and 6.0.

[0043] Also described herein are kits comprising one or more of the antibodies described herein in a suitable container and one or more additional components selected from: instructions for use; a diluent, an excipient, a carrier, and a device for administration.

[0044] In certain embodiments, described herein is a method of preparing a cancer treatment comprising admixing one or more pharmaceutically acceptable excipients, carriers, or diluents and an antibody of the current disclosure. In certain embodiments, described herein is a method of preparing a cancer treatment for storage or shipping comprising lyophilizing one or more antibodies of the current disclosure. Anti-RORl Antibodies

[0045] The pharmaceutical compositions described and provided herein comprise the anti-RORl antibodies or ROR1 -binding antibody fragments described herein.

Bruton ’s tyrosine kinase (BTK) Inhibitors

[0046] Bruton’s tyrosine kinase (BTK) is a non-receptor kinase that plays an important role in oncogenic signaling. BTK has been identified as a critical factor for proliferation and survival of leukemic cells in hematologic cancers. Inhibitors of BTK have been developed for cancer treatment. One BTK inhibitor, ibrutinib, was used as first-line treatment for patients with B cell malignancies. By disrupting B-cell signaling pathways, BTK treatment has been associated with a dramatic lymph node response, but eradication of disease and relapse in high-risk disease remain challenges. In some cases, cancers are resistant to treatment with BTKIs. Especially for patients with TP53 mutation, the outcome of BTK treatment still needs to be improved.

[0047] The BTK inhibitor (BTKi) disclosed herein which the cancer is resistant to can be a small molecule. In some instances, the BTKi is ibrutinib, acalabrutinib, evobrutinib, fenebrutinib, orelabrutinib, rilzabrutinib, tolebrutinib, branebrutinib, nemtabrutinib, elsubrutinib, remibrutinib, vecabrutinib, remibrutinib, spebrutinib, tirabrutinib, AC0058TA, pirtobrutinib (LOXO-305), zanubrutinib, spebrutinib, DTRMWXHS-12, M7583, SN-1011, TG-1701, or combinations thereof. In some instances, the BTKi is ibrutinib. In some instances, the BTKi is acalabrutinib. In some instances, the BTKi is tirabrutinib (ONO/GS- 4059). In some instances, the BTKi is zanubrutinib (BGB-3111). In some instances, the BTKi is CC-292. In some instances, the BTKi is spebrutinib (AVL-292).

B-cell lymphoma 2 (BCL2) Inhibitors

[0048] Bcl-2 (B-cell lymphoma 2), encoded in humans by the BCL2 gene, is the founding member of the Bcl-2 family of regulator proteins that regulate cell death (apoptosis), by either inhibiting (anti-apoptotic) or inducing (pro-apoptotic) apoptosis. Damage to the Bcl-2 gene has been identified as a cause of a number of cancers, including melanoma, breast, prostate, chronic lymphocytic leukemia, and lung cancer. It is also a cause of resistance to cancer treatments. Defects in the apoptosis mechanism stimulate cancer cell growth and survival. B cell lymphoma 2 (Bcl-2) is an anti-apoptotic molecule that plays a central role in apoptosis. Bcl-2 has been identified as being over-expressed in several cancers. Bcl-2 is induced by protein kinases and several signaling molecules which stimulate cancer development. Because of the fundamental function of Bcl-2 in the regulation of apoptosis, the Bcl-2 protein is a potent target for the development of novel anti-tumor treatments. In some cases, cancers are resistant to treatment with Bcl-2 inhibitors. In some instances, the Bcl-2 inhibitor is venetoclax, navitoclax, ABT-737, AZD5991, AMG176, AMG397, S64315, ABBV467, or combinations thereof. In some cases, cancers are resistant to treatment with Bcl-2 inhibitors.

Compositions

[0049] Provided herein are compositions (e.g., pharmaceutical compositions) comprising: an anti-RORl antibody or ROR1 -binding antibody fragment thereof; and about 5% (w/v) or greater trehalose; wherein the anti-RORl antibody or ROR1 -binding antibody fragment thereof comprises:

(a) a heavy chain complementarity determining region 1 (H-CDR1) comprising an amino acid sequence set forth in SEQ ID NO: 1 ;

Further provided are compositions (e.g., pharmaceutical compositions) comprising: an anti-RORl antibody or ROR1 -binding antibody fragment thereof; and about 5% (w/v) or greater trehalose; wherein the anti-RORl antibody or ROR1 -binding antibody fragment thereof comprises:

(a) a heavy chain complementarity determining region 1 (H-CDR1); (b) a heavy chain complementarity determining region 2 (H-CDR2);

(c) a heavy chain complementarity determining region 3 (H-CDR3);

(d) a light chain complementarity determining region 1 (L-CDR1);

(e) a light chain complementarity determining region 2 (L-CDR2); and/or

(f) a light chain complementarity determining region 3 (L-CDR3); wherein the H-CDR1, the H-CDR2, and the H-CDR3 are derived from SEQ ID NO: 7, and the L-CDR1, the L-CDR2, and the L-CDR3 are derived from SEQ ID NO: 8; and wherein the H-CDR1, the H-CDR2, the H-CDR3, L-CDR1, the L-CDR2, and the L-CDR3 are defined using Chothia, Kabat, IMGT, Contact, or AbM methodologies. In some embodiments, the anti-RORl antibody or ROR1 -binding antibody fragment thereof comprises: a heavy chain variable domain comprising an amino acid sequence having 70% or greater sequence identity to SEQ ID NO: 7; and a light chain variable domain comprising an amino acid sequence having 70% or greater sequence identity to SEQ ID NO: 8.

In some embodiments, the anti-RORl antibody comprises: a heavy chain domain comprising an amino acid sequence having 70% or greater sequence identity to SEQ ID NO: 9; and a light chain domain comprising an amino acid sequence having 70% or greater sequence identity to SEQ ID NO: 10.

In some embodiments, the anti-RORl antibody comprises: a heavy chain constant domain comprising an amino acid sequence having 70% or greater sequence identity to SEQ ID NO: 11.

[0050] Further provided are compositions (e.g., pharmaceutical compositions) comprising: an anti-RORl antibody or ROR1 -binding antibody fragment thereof; and about 5% (w/v) or greater trehalose; wherein the anti-RORl antibody or ROR1 -binding antibody fragment thereof comprises: a heavy chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO: 7; and a light chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO: 8. [0051] Also provided herein are compositions (e.g., pharmaceutical compositions) comprising: an anti-RORl antibody; and about 5% (w/v) or greater trehalose; wherein the anti-RORl antibody comprises: a heavy chain domain comprising an amino acid sequence as set forth in SEQ ID NO: 9; and a light chain domain comprising an amino acid sequence as set forth in SEQ ID NO: 10.

Further provided are compositions (e.g., pharmaceutical compositions) comprising zilovertamab and 5% (w/v) or greater trehalose;

[0052] In some embodiments, the composition comprises the anti-RORl antibody or ROR1 -binding antibody fragment thereof at a concentration of between about 20 mg/mL to about 80 mg/mL. In some embodiments, the composition comprises the anti-RORl antibody or ROR1 -binding antibody fragment thereof at a concentration of between about 20 mg/mL to about 60 mg/mL. In some embodiments, the composition comprises the anti-RORl antibody or ROR1 -binding antibody fragment thereof at a concentration of between about 30 mg/mL to about 50 mg/mL. In some embodiments, the composition comprises the anti-RORl antibody or ROR1 -binding antibody fragment thereof at a concentration of between about 40 mg/mL to about 50 mg/mL. In some embodiments, the composition comprises the anti-RORl antibody or ROR1 -binding antibody fragment thereof at a concentration of between about 20 mg/mL to about 80 mg/mL. In some embodiments, the composition comprises the anti-RORl antibody or ROR1 -binding antibody fragment thereof at a concentration of about 40 mg/mL. In some embodiments, the composition comprises the anti-RORl antibody or ROR1 -binding antibody fragment thereof at a concentration of about 100 ng/mL.

[0054] In some embodiments, the composition further comprises a buffer (e.g., 5-20 mM). In certain embodiments, the buffer is a citrate buffer (e.g., sodium citrate). In certain embodiments, the composition comprises between about 5 mM to about 20 mM citrate buffer. In certain embodiments, the composition comprises less than 20 mM citrate buffer. In certain embodiments, the composition comprises less than 15 mM citrate buffer. In certain embodiments, the composition comprises about 10 mM citrate buffer.

[0055] In some embodiments, the composition is buffered to comprise a pH between about 4.0 to about 6.0. In certain embodiments, the composition is buffered to comprise a pH between about 4.5 to about 6.0. In certain embodiments, the composition is buffered to comprise a pH between about 5.0 to about 6.0. In certain embodiments, the composition is buffered to comprise a pH between about 4.5 to about 5.5. In certain embodiments, the composition is buffered to comprise a pH between about 5.0 to about 5.5. In certain embodiments, the composition is buffered to comprise a pH of about 5.2.

[0056] In some embodiments, the composition further comprises a surfactant (e.g., 0.005- 0.100% (w/v)). In certain embodiments, the surfactant comprises a non-ionic surfactant (e.g., 0.005-0.100% (w/v)). In certain embodiments, the surfactant comprises polysorbate 80. In certain embodiments the composition comprises about 0.005% (w/v) to about 0.100% (w/v) polysorbate 80. In certain embodiments the composition comprises about 0.010% (w/v) to about 0.080% (w/v) polysorbate 80. In certain embodiments the composition comprises about 0.010% (w/v) to about 0.050% (w/v) polysorbate 80. In certain embodiments the composition comprises about 0.01% (w/v) to about 0.030% (w/v) polysorbate 80. In certain embodiments the composition comprises about 0.015% (w/v) to about 0.025% (w/v) polysorbate 80. In certain embodiments the composition comprises about 0.020% (w/v) polysorbate 80.

[0057] In some embodiments, the composition further comprises a chelator (e.g., 0.01- 0.10 mM). In some embodiments, the chelator comprises EDTA. In certain embodiments, the composition comprises about 0.001 mM to about 0.350 mM EDTA. In certain embodiments, the composition comprises about 0.010 mM to about 0.100 mM EDTA. In certain embodiments, the composition comprises about 0.020 mM to about 0.080 mM EDTA. In certain embodiments, the composition comprises about 0.040 mM to about 0.060 mM EDTA. In certain embodiments, the composition comprises about 0.050 mM EDTA.

[0058] In some embodiments, the composition comprises: the anti-RORl or ROR1 -binding antibody fragment thereof at a concentration of about 40 milligrams per milliliter; about 10 mM sodium citrate; about 0.05 mM EDTA; about 0.02% (w/v) polysorbate 80; and about 7.5% (w/v) trehalose (e.g., trehalose-2H2O); wherein the pH of the composition is about 5.2.

[0059] In some embodiments, the composition is a for use in an intravenous injection. In some embodiments, the composition is administered intravenously. Other injection routes can be contemplated, for example, subcutaneous, intraperitoneal, intramuscular, intratumoral, or intracerebral, etc.

Methods

[0061] Provided herein are methods of treating cancer or a tumor, the method comprising: administering the compositions described herein comprising the ROR1 antibody or ROR1 -binding fragment thereof to an individual in need thereof. For example, provided methods of treating cancer or a tumor, the method comprising: administering a composition comprising an anti-RORl antibody or ROR1- binding antibody fragment thereof; and about 5% (w/v) or greater trehalose; wherein the anti-RORl antibody or ROR1 -binding antibody fragment thereof comprises:

(b) a heavy chain complementarity determining region 2 (H-CDR2) comprising an amino acid sequence set forth in SEQ ID NO: 2; (c) a heavy chain complementarity determining region 3 (H-CDR3) comprising an amino acid sequence set forth in SEQ ID NO: 3;

[0062] In some embodiments, the cancer is a leukemia or a lymphoma. In certain embodiments, the leukemia or lymphoma is a B cell leukemia or lymphoma. In certain embodiments, the leukemia or lymphoma is chronic lymphocytic leukemia (CLL). In certain embodiments, the leukemia or lymphoma is mantle cell lymphoma (MCL). In certain embodiments, the leukemia or lymphoma is small cell lymphocytic leukemia (SLL). In certain embodiments, the leukemia or lymphoma is marginal zone lymphoma (MZL). In some embodiments, the cancer is a solid tissue cancer and/or comprises a solid tumor. In certain embodiments, the solid tissue cancer is breast cancer, ovarian cancer, prostate cancer, lung cancer, pancreatic cancer, head and neck cancer, kidney cancer, colon cancer, or stomach cancer. In some embodiments, administering the ROR1 binding antibody or the ROR1 antigen binding fragment induces antibody dependent cellular cytotoxicity of the cancer. In some embodiments, the ROR1 binding antibody or the ROR1 antigen binding fragment thereof exhibits antibody dependent cellular cytotoxicity which is greater than or equal to antibody dependent cellular cytotoxicity exhibited by rituximab. In some embodiments, the antibody dependent cellular cytotoxicity is exhibited against a leukemic cell. In some embodiments, the leukemic cell comprises a CLL cells, MZL, or MCL cell.

Patient Populations

[0064] The cancer or tumor comprises at least one mutation in TP53. In some instances, the mutation is in an exon of the TP53 gene. In some instances, the mutation is in an intron of the TP53 gene. In some instances, the mutation is a nonsense mutation in the TP53 gene. In some instances, the mutation is a missense mutation in the TP53 gene. In some instances, the mutation results in an amino acid insertion, deletion, or substitution in a protein encoded by the TP53 gene. In some instances, the mutation is associated with del(17p). In some instances, the mutation is a splice-site mutation. In some instances, the mutation is in at least one exon from exons 4-8 of TP53. For example, the mutation can be in exon 4, exon 5, exon 6, exon 7, or exon 8 of TP53. In some instances, the mutation results in an aberration in the DNA-binding domain of p53. In some instances, the mutation results in an aberration in the oligomerization domain of p53. In some instances, the mutation results in an aberration in the C-terminal domain of p53. In some instances, the mutation comprises a single mutation as described herein or a combination of the single mutations as described herein. For example, the mutation can comprise a missense mutation and a del(17p) mutation. The mutation can comprise an insertion mutation and a del(17p) mutation. The mutation can comprise a gene deletion mutation and a del(17p) mutation. The mutation can comprise a splice-site mutation and a del(17p) mutation. The mutation can comprise an insertion mutation and a missense mutation. The mutation can comprise a gene deletion mutation and a missense mutation. The mutation can comprise a splice-site mutation and a missense mutation. The mutation can comprise an insertion mutation and a gene deletion mutation. The mutation can comprise an insertion mutation and a splice-site mutation. The mutation can comprise a gene deletion mutation and a splice-site mutation. The mutation in TP53 can comprise any mutation that results in a loss of wildtype p53 function in a cell.

[0065] The subject may be screened for ROR1 expression before, at the time of, or after administration of the BTKi and ROR1 antagonist. The subject may be further subject to a TP53 mutation screening before, at the time of, or after administration of the BTKi and ROR1 antagonist. In some instances, the methods disclosed herein comprises determining if the tumor or cancer comprises a mutation in the TP53 gene. A test can be performed on the tumor or the cancer to determine if the tumor or cancer comprises a mutation in the TP53 gene. The test can be performed on a biopsy sample of the tumor or the cancer of the subject. The test can be based either on genomic DNA or mRNA as template. The test can also be based on p53 protein. For example, the test can be immunohistochemistry, immunofluorescence, PCR, RT-PCR, real-time PCR, fluorescence in situ hybridization (FISH), sequencing including next generation sequencing, or genomic arrays.

[0066] In some instances, the subject is a mammal. In some instances, the subject is a human. In some instances, the subject having a tumor or cancer with a TP53 mutation. In some instances, the subject having a tumor or cancer with a TP53 mutation and ROR1 expression. In some instances, the subject is a relapsed patient from prior treatment of the cancer or tumor. In some instances, the subject is a patient who developed resistance to prior treatment of the cancer or tumor.

Nucleic Acids

[0068] The anti-RORl antibodies described herein can be encoded by a nucleic acid. A nucleic acid is a type of polynucleotide comprising two or more nucleotide bases. In certain embodiments, the nucleic acid is a component of a vector that can be used to transfer the polypeptide encoding polynucleotide into a cell. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a genomic integrated vector, or “integrated vector,” which can become integrated into the chromosomal DNA of the host cell. Another type of vector is an “episomal” vector, e.g., a nucleic acid capable of extra-chromosomal replication. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors.” Suitable vectors comprise plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors and the like. In the expression vectors regulatory elements such as promoters, enhancers, polyadenylation signals for use in controlling transcription can be derived from mammalian, microbial, viral or insect genes. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants may additionally be incorporated. Vectors derived from viruses, such as lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, and the like, may be employed. Plasmid vectors can be linearized for integration into a genome.

[0069] In certain embodiments, described herein, is a master cell bank comprising: (a) a mammalian cell line comprising a nucleic acid encoding an antibody described herein integrated at a genomic location; and (b) a cryoprotectant. In certain embodiments, the cryoprotectant comprises glycerol or DMSO. In certain embodiments, the master cell bank comprises: (a) a CHO cell line comprising a nucleic acid encoding an antibody with (i) a heavy chain amino acid sequence set forth by any one of SEQ ID NOs: 1, 2 or 3; and (ii) a light chain amino acid sequence set forth by any one of SEQ ID NOs: 4, 6, or sequence SGS integrated at a genomic location; and (b) a cryoprotectant. In certain embodiments, the master cell bank comprises: (a) a CHO cell line comprising a nucleic acid encoding an antibody with an amino acid sequence set forth SEQ ID NOs: 7, 8, 9, or 10. In certain embodiments, the cryoprotectant comprises glycerol or DMSO. In certain embodiments, the master cell bank is contained in a suitable vial or container able to withstand freezing by liquid nitrogen.

[0070] Also described herein are methods of making an anti-RORl antibody described herein. Such methods comprise incubating a cell or cell-line comprising a nucleic acid encoding the anti-RORl antibody in a cell culture medium under conditions sufficient to allow for expression and secretion of the antibody, and further harvesting the anti-RORl antibody from the cell culture medium. The harvesting can further comprise one or more purification steps to remove live cells, cellular debris, non-antibody proteins or polypeptides, undesired salts, buffers, and medium components. In certain embodiments, the additional purification step(s) include centrifugation, ultracentrifugation, protein A, protein G, protein A/G, or protein L purification, and/or ion exchange chromatography.

Definitions

[0072] An antibody is used in the broadest sense, and generally encompasses and/or refers to various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. In some embodiments, an antibody or antibodies include intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen binding (Fab) fragments, F(ab’)2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (sFv or scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. In some embodiments, an antibody or antibodies include genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. In some embodiments, an antibody or antibodies encompass functional antibody fragments thereof. In some embodiments, an antibody or antibodies encompasses intact or full- length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD. The antibody can comprise a human IgGl constant region. The antibody can comprise a human IgG4 constant region. In some embodiments, an antibody or antibodies include, but is not limited to, full-length and native antibodies, as well as fragments and portions thereof retaining the binding specificities thereof, such as any specific binding portion thereof including those having any number of, immunoglobulin classes and/or isotypes (e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE and IgM); and biologically relevant (antigen-binding) fragments or specific binding portions thereof, including but not limited to Fab, F(ab’)2, Fv, and scFv (single chain or related entity). A monoclonal antibody is generally one within a composition of substantially homogeneous antibodies; thus, any individual antibodies comprised within the monoclonal antibody composition are identical except for possible naturally occurring mutations that can be present within a composition in minor amounts. A monoclonal antibody can comprise a human IgGl constant region or a human IgG4 constant region.

[0073] A native antibody generally encompasses and/or refers to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies can be monomeric or multimeric glycoproteins, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3). Similarly, from N- to C- terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa (K) and lambda (X), based on the amino acid sequence of its constant domain.

[0074] A full-length antibody, intact antibody, and whole antibody are interchangeable, and generally include and/or refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein. [0075] A human consensus framework generally encompasses and/or refers to a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In some embodiments, for the VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In some embodiments, for the VH, the subgroup is subgroup III as in Kabat et al., supra. A humanized antibody encompasses and refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human framework regions (FRs). In some embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally encompasses at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0076] A complementarity determining region or CDR, which are synonymous with hypervariable region or HVR, generally include and/or refer to regions of an antibody which are hypervariable in sequence and/or form structurally defined loops (hypervariable loops) and/or contain the antigen-contacting residues (antigen contacts). In some embodiments, complementarity determining regions or CDRs generally include and refer to non-contiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and/or binding affinity. In general, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3).

[0077] A framework region or FR generally includes and/or refers to the non-CDR portions of the variable regions of the heavy and light chains. In general, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4). In some embodiments, the framework regions are defined by the non-CDR sequences of a variable region sequence. The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745.” (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 Jan;27(l):55-77 (“IMGT” numbering scheme); Honegger A and Pliickthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8;309(3):657-70, (“Aho” numbering scheme); and Whitelegg NR and Rees AR, “WAM: an improved algorithm for modelling antibodies on the WEB,” Protein Eng. 2000 Dec;13(12):819-24 (“AbM” numbering scheme. In certain embodiments, the CDRs of the antibodies described herein can be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or combinations thereof. In some embodiments, the CDRs and FRs are defined by and/or according to a Kabat numbering scheme. In some embodiments, the CDRs and FRs are defined by and/or according to a Chothia numbering scheme. In some embodiments, the CDRs and FRs are defined by and/or according to a IMGT numbering scheme. In some embodiments, the CDRs and FRs are defined by and/or according to an EU numbering scheme.

[0078] The boundaries of a given CDR or FR, in certain instances, vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural 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,” and deletions appearing in some antibodies. 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.

[0079] The Fc region of an antibody generally encompasses and/or refers to a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In some embodiments, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0080] A human antibody generally encompasses and/or refers to an antibody which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. [0081] An acceptor human framework region generally encompasses and/or refers to a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence thereof, or it can contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

[0082] A variable region or variable domain generally encompasses and/or refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs (See e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91(2007)). A single VH or VL domain can be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen can be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively See e.g, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)). [0083] Affinity generally encompasses and/or refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, binding affinity generally encompasses and refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described throughout.

[0084] An affinity matured antibody generally encompasses and/or refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.

[0085] Binding and a determination of binding can be readily determined by methods known within the art (e.g., ELISA, surface plasmon resonance, bio-layer interferometry, isothermal calorimetry, etc.). In some embodiments, binding is determined by ELISA. In some embodiments, binding comprising a KD less than, e.g., 10^A-5 M (lOuM) as measured by surface plasmon resonance, bio-layer interferometry, or isothermal calorimetry. In some embodiments, binding comprising a Ko less than, e.g., 10^A-6 M (luM) surface plasmon resonance, bio-layer interferometry, or isothermal calorimetry. In some embodiments, binding comprising a Koless than, e.g., 10^A-7 M (lOOnM) surface plasmon resonance, biolayer interferometry, or isothermal calorimetry.

[0086] A chimeric antibody generally encompasses and/or refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species. 1 The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgGi, IgG?, IgGs, IgG₄, IgAi, and IgA?. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively.

[0087] A monoclonal antibody generally encompasses and/or refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present within a composition in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In some embodiments, the modifier monoclonal indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present within a composition disclosure can be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

[0088] An isolated antibody generally encompasses and/or refers to an antibody that has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC).

[0089] In certain embodiments, the antibody comprises one or more naturally occurring amino acids. In certain embodiments, the antibody consists of naturally occurring amino acids. As used herein, naturally occurring amino acids include and/or refer to amino acids which are generally found in nature and are not manipulated by man. In certain instances, naturally occurring includes and/or further refers to the 20 conventional amino acids: alanine (A or Ala), cysteine (C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H or His), isoleucine (I or He), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), glutamine (Q or Gin), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Vai), tryptophan (W or Trp), and tyrosine (Y or Tyr).

[0090] In some embodiments, the antibody comprises a variant sequence of the antibody. In certain instances, amino acid substitutions can be made in the sequence of any of the antibodies described herein, without necessarily decreasing or ablating its activity (as measured by, e.g., the binding or functional assays described herein). Accordingly, in some embodiments, the variant sequence comprises one or more amino acid substitutions. In some embodiments, the variant sequence comprises one or more substitutions in one or more CDRs. In certain embodiments, the variant sequence comprises one amino acid substitution. In certain embodiments, the variant sequence comprises two amino acid substitutions. In certain embodiments, the variant sequence comprises three amino acid substitutions. In certain instances, substitutions include conservative substitutions (e.g., substitutions with amino acids of comparable chemical characteristics). In certain instances, a non-polar amino acid can be substituted and replaced with another non-polar amino acid, wherein non-polar amino acids include alanine, leucine, isoleucine, valine, glycine, proline, phenylalanine, tryptophan and methionine. In certain instances, a neutrally charged polar amino acids can be substituted and replaced with another neutrally charged polar amino acid, wherein neutrally charged polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. In certain instances, a positively charged amino acid can be substituted and replaced with another positively charged amino acid, wherein positively charged amino acids include arginine, lysine and histidine. In certain instances, a negatively charged amino acid can be substituted and replaced with another negatively charged amino acid, wherein negatively charged amino acids include aspartic acid and glutamic acid. Examples of amino acid substitutions also include substituting an L-amino acid for its corresponding D-amino acid, substituting cysteine for homocysteine or other non-natural amino acids.

[0091] In certain embodiments, the antibody comprises one or more non-natural amino acids. In certain embodiments, the antibody consists of non-natural amino acids. As used herein, non-natural amino acids and/or unnatural amino acids include and/or refer to amino acid structures that cannot be generated biosynthetically in any organism using unmodified or modified genes from any organism. For example, these include, but are not limited to, modified amino acids and/or amino acid analogues that are not one of the 20 naturally occurring amino acids (e.g., non-natural side chain variant sequence amino acids), D-amino acids, homo amino acids, beta-homo amino acids, N-methyl amino acids, alpha-methyl amino acids, or. By way of further example, non-natural amino acids also include 4- Benzoylphenylalanine (Bpa), Aminobenzoic Acid (Abz), Aminobutyric Acid (Abu), Aminohexanoic Acid (Ahx), Aminoisobutyric Acid (Aib), Citrulline (Cit), Diaminobutyric Acid (Dab), Diaminopropanoic Acid (Dap), Diaminopropionic Acid (Dap), GammaCarb oxy glutamic Acid (Gia), Homoalanine (Hala), Homoarginine (Harg), Homoasparagine (Hasn), Homoaspartic Acid (Hasp), Homocysteine (Heys), Homoglutamic Acid (Hglu), Homoglutamine (Hgln), Homoisoleucine (Hile), Homoleucine (Hleu), Homomethionine (Hmet), Homophenylalanine (Hphe), Homoserine (Hser), Homotyrosine (Htyr), Homovaline (Hval), Hydroxyproline (Hyp), Isonipecotic Acid (Inp), N aphthylalanine (Nal), Nipecotic Acid (Nip), Norleucine (Nle), Norvaline (Nva), Octahydroindole-2-carboxylic Acid (Oic), Penicillamine (Pen), Phenylglycine (Phg), Pyroglutamic Acid (Pyr), Sarcosine (Sar), tButylglycine (Tie), and Tetrahydro-isoquinoline-3-carboxylic Acid (Tic). Such non-natural amino acid residues can be introduced by substitution of naturally occurring amino acids, and/or by insertion of non-natural amino acids into the naturally occurring antibody sequence. A non-natural amino acid residue also can be incorporated such that a desired functionality is imparted to the apelin molecule, for example, the ability to link a functional moiety (e.g., PEG).

[0092] ROR1 generally refers to and includes a human ROR1 protein or gene encoding a ROR1 protein (synonyms: tyrosine-protein kinase transmembrane receptor ROR1, EC=2.7.10.1, neurotrophic tyrosine kinase, receptor-related 1, UniPrtKB Q01973), which is a tyrosine-protein kinase receptor. The extracellular domain of ROR1 consists according to of amino acids 30-406.

[0093] An antibody against ROR1 or anti-RORl antibody generally refers to and includes an antibody that specifically binds to human ROR1 (e.g., ELISA, surface plasmon resonance, bio-layer interferometry, isothermal calorimetry, etc.). In certain embodiments, the antibody binds specifically to the extracellular domain of ROR1. In certain embodiments, the antibody binds specifically to fragments of the extracellular domain, which are the Ig-like C2-type domain, the frizzled domain, or the kringle domain. These fragments are mentioned in W02005100605. It is further disclosed that the antibody may bind specifically to the extracellular domain fragment WNISSELNKDSYLTL of ROR1. This fragment is disclosed in Daneshmanesh A H et al., Int. J. Cancer, 123 (2008) 1190-1195.

[0094] As used herein an ROR1 binding antibody or an ROR1 antigen binding fragment thereof can refer to an ROR1 binding antibody or the ROR1 antigen binding fragment thereof that comprises an alteration to increase antibody effector function antibody, or dependent cell cytotoxicity (ADCC). The alteration may include glycoengineering, glycosylation, deglycosylation, or afucosylation.

[0095] A pharmaceutically acceptable carrier generally encompasses and/or refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier encompasses, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0096] A polypeptide or protein are used interchangeably, and generally encompass and/or refer to a polymer of amino acid residues, and are not limited to a minimum length. Polypeptides, including the provided antibodies and antibody chains and other peptides, e.g., linkers and binding peptides, can include amino acid residues including natural and/or nonnatural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some embodiments, the polypeptides can contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications can be deliberate, as through site-directed mutagenesis, or can be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0097] R0R1 expressing cancer or R0R1 expressing cancer cell generally refers to and includes to all neoplastic cell growth and proliferation, whether malignant or benign, including all transformed cells and tissues and all cancerous cells and tissues, that express, over-express, or abnormally express ROR1.

[0098] The determination of percent identity or percent similarity between two sequences can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-410. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, and PSI- Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti, 1994, Comput. AppL Biosci. 10:3-5; and FASTA described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-8. Alternatively, sequence alignment may be carried out using the CLUSTAL algorithm (e.g., as provided in the program Clustal- omega), as described by Higgins et al., 1996, Methods EnzymoL 266:383-402.

[0099] As used herein the term individual, patient, or subject generally includes and/or refers to individuals diagnosed with, suspected of being afflicted with, or at-risk of developing at least one disease, condition, or status for which the described compositions and method are useful for treating. In certain embodiments, the individual is a mammal. In certain embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. In certain embodiments, the individual is a human.

[00100] As used herein, treatment or treating generally include and/or refer to a pharmaceutical or other intervention regimen used for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and/or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made. Skilled artisans will recognize that given a population of potential individuals for treatment not all will respond or respond equally to the treatment. Such individuals are considered treated.

[00101] As used herein, the words modulation and regulation can be used interchangeably herein. In some embodiments, modulation comprises a decrease and/or reduction and/or inhibition. In some embodiments, modulation comprises to an increase and/or induction and/or promotion and/or activation.

[00102] As used herein, the words comprising (and any form of comprising, such as comprise and comprises), having (and any form of having, such as have and has), including (and any form of including, such as include and includes) or containing (and any form of containing, such as contain and contains), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps. As also used herein, in any instance or embodiment described herein, comprising may be replaced with consisting essentially of and/or consisting of. As used herein, in any instance or embodiment described herein, comprises may be replaced with consists essentially of and/or consists of.

[00103] As used herein, the term about in the context of a given value or range includes and/or refers to a value or range that is within 10% of the given value or range.

[00104] As used herein, the term and/or is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, A and/or B is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each were set out individually herein.

EXAMPLES

[00105] The following illustrative examples are representative of embodiments of the compositions and methods described herein and are not meant to be limiting in any way.

Example 1. Manufactured Antibody Zilovertamab contains no fucose in the Fc region

[00106] The antibody Zilovertamab was manufactured and its glycosylation was further characterized by liquid chromatography-mass spectrometry (LC-MS). IgG antibody was digested with FabRICATOR protease in PBS supplemented with DTT. The protease cuts IgG antibodies at one specific site below the hinge which, together with reduction of intermolecular disulfide bonds by DTT, yields three subunits (scFc, LC and Fd’). These subunits were analyzed further by reverse-phase LC-MS on a Waters BioAccord LC-MS System equipped with a Waters BioResolve RP mAb column (2.1x50mm). The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn 297, relative to the sum of all glycostructures attached to Asn297. However, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. The Fc glycosylation profile of recombinantly expressed IgGs, including most approved therapeutic mAbs, is generally dominated by G0F and GIF structures with lower amounts of G2F and afucosylated GO. The analysis of glycosylation of zilovertamab indicates that the manufactured zilovertamab contains no fucose at GO or G1 structure (Table 1).

Example 2. Development And Testing Of A Glycoengineered Anti-RORl Antibody With Enhanced Capacity For Directing Antibody-Dependent Cellular Cytotoxicity (ADCC) Of Chronic Lymphocytic Leukemia Cells [00107] Receptor tyrosine kinase-like orphan receptor 1 (R0R1) is an oncoembryonic surface antigen expressed on the neoplastic cells of patients with chronic lymphocytic leukemia (CLL) or other types of cancer, but not on virtually all normal postpartum tissues (Fukuda T. et al., Proc. Natl. Acad. Sci. USA, 105:3047, 2008). Zilovertamab (also known as UC-961 or cirmtuzumab) is a humanized anti-RORl monoclonal antibody (mAb) currently under clinical investigation; this mAb is capable of blocking ROR1 -signaling, which can contribute to cancer-cell migration, proliferation, survival, and self-renewal.

[00108] A glycoengineered afucosylated form of zilovertamab (GE-zilovertamab) was generated according to the methodology as previously described, and examined for whether it could mediate enhanced antibody-dependent cellular cytotoxicity (ADCC) against neoplastic cells that express ROR1. Initial studies used Jurkat-Lucia™ NFAT-CD16 cells (Invivogen, San Diego, CA, USA), which express high-affinity CD16A (FcyRIIIA, VI 58 allotype) as effector cells (EC), and the CLL-cell line MEC1, or MEC1 cells transduced to express ROR1 (MEC1-ROR1), as target cells (TC), which each also express CD20. Jurkat-Lucia effector cells were co-cultured with MEC1 or MEC1-ROR target cells at EC:TC ratio of 10:1 for 6 hours at 37 °C with or without anti-CD20 mAb rituximab or anti- ROR mAbs, such as zilovertamab or glycoengineered afucosylated-zilovertamab (GE- zilovertamab) cells, at concentrations of 100 ng/ml. Luminescence activity was measured after 6 hours of co-culture to demonstrate activation of Jurkat-Lucia cells.

[00109] FIG. 1 shows activation of Jurkat-Lucia NFAT-CD16 cells by cells treated with anti-CD20 or anti-RORl mAb. Neither zilovertamab nor GE-zilovertamab endowed Jurkat- Lucia cells with higher than background luminescence activity when co-cultured with MEC1 cells. However, GE-zilovertamab could induce high levels of luminescence activity in Jurkat- Lucia cells co-cultured with MEC1-ROR1 cells that was comparable to that of rituximab, and significantly greater than that of zilovertamab (P<0.05).

[00110] In another condition, Jurkat-Lucia effector cells were co-cultured with MEC1 or MEC1-ROR target cells at EC:TC ratio of 20: 1 for 6 hours at 37 °C with or without anti- CD20 mAb rituximab or anti-ROR mAbs, such as zilovertamab or GE-zilovertamab cells, at concentrations of 100 ng/ml. Luminescence activity was measured after 6 hours of co-culture to demonstrate activation of Jurkat-Lucia cells.

[00111] FIG. 2 shows activation of Jurkat-Lucia NFAT-CD16 cells by cells treated with anti-CD20 or anti-RORl mAb. Neither zilovertamab nor GE-zilovertamab endowed Jurkat- Lucia cells with higher than background luminescence activity when co-cultured with MEC1 cells. However, GE-zilovertamab could induce high levels of luminescence activity in Jurkat- Lucia cells co-cultured with MEC1-R0R1 cells that was comparable to that of rituximab, and significantly greater than that of zilovertamab (P<0.01).

[00112] Jurkat-Lucia effector cells were co-cultured with MEC1 or MEC1-ROR target cells at EC:TC ratio of 30: 1 for 6 hours at 37 °C with or without anti-CD20 mAb rituximab or anti-ROR mAbs, such as zilovertamab or GE-zilovertamab cells, at concentrations of 100 ng/ml. Luminescence activity was measured after 6 hours of co-culture to demonstrate activation of Jurkat-Lucia cells.

[00113] FIG. 3 shows activation of Jurkat-Lucia NFAT-CD16 cells by cells treated with anti-CD20 or anti-RORl mAb. Neither zilovertamab nor GE-zilovertamab endowed Jurkat- Lucia cells with higher than background luminescence activity when co-cultured with MEC1 cells. However, GE-zilovertamab could induce high levels of luminescence activity in Jurkat- Lucia cells co-cultured with MEC1-ROR1 cells that was comparable to that of rituximab, and significantly greater than that of zilovertamab.

[00114] An orthogonal system was generated and used to examine the relative ADCC activity of each of these mAb, using effector cells (EC) generated from the NK cell line, NK92, which was transduced with a lentivirus encoding a newly-generated high-affinity variant of CD16A (CD16v), which stably expresses the cell surface Fc receptor CD16A (FcyRIIIA; VI 76 and Pl 97 mutated). NK92 or NK92-CD16v were used as EC to effect cytolysis of Cr⁵¹-labeled MEC1 or MEC1-ROR1 target cells (TC) in a chromium-release assay at EC:TC ratio of 2: 1 for 6 hours at 37 °C with or without anti-CD20 mAb rituximab or anti-ROR mAbs, such as zilovertamab or GE-zilovertamab, at concentrations of 100 ng/ml. Chromium (Cr⁵¹) release was measured after 6-hours of co-culture and percent cell lysis was calculated relative to trichloroacetic acid (TCA) treated cells.

[00115] FIG. 4 shows ADCC by NK92-CD16v cells for target cells treated with anti- RORl or anti-CD20 mAb. Addition of rituximab to co-cultures of EC NK92-CD16v with Cr⁵¹-labeled MEC1 or MEC1-ROR1 TC, both of which express CD20, caused mAb-dose- dependent ADCC of each of these TC by NK92-CD16v that was significantly greater than that by parental NK92 cells. Neither zilovertamab nor GE-zilovertamab could direct ADCC by NK92-CD16v of MEC1 cells that lack ROR1 expression. However, GE-zilovertamab directed dose-dependent ADCC of MEC1-ROR1 by either NK92-CD16v at levels comparable to that directed by rituximab, and such levels were significantly greater than that directed by zilovertamab (P<0.05).

[00116] FIG. 5 shows ADCC by NK92-CD16v cells for ROR1+ CLL cells treated with anti-RORl or anti-CD20 mAb. NK92 or NK92-CD16v were used as EC to effect cytolysis of Cr⁵¹-labeled primary patient-derived ROR+ CLL TC cells in a chromium-release assay at EC:TC TC ratio of 10: 1 for 6 hours at 37 °C with or without anti-CD20 mAh rituximab or anti-ROR mAbs, such as zilovertamab or GE-zilovertamab, at concentrations of 100 ng/ml. Chromium (Cr⁵¹) release was measured after 6-hours of co-culture and percent specific cell lysis was calculated relative to trichloroacetic acid (TCA) treated cells.

[00117] Addition of GE-zilovertamab to co-cultures of EC NK92-C16 with Cr⁵¹-labeled patient-derived primary R0R+ CLL TC, which expresses both CD20 and R0R1, caused m Ab-dose-dependent ADCC of TC by NK92-CD16v at levels comparable to that directed by rituximab. Such levels were significantly greater than that directed by zilovertamab (P<0.05). [00118] FIG. 6 shows ADCC by NK92 or NK92-CD16v cells for R0R1+ CLL cells treated with anti-RORl or anti-CD20 mAb. NK92 or NK92-CD16v cells were used as EC to effect cytolysis of Cr⁵¹-labeled primary patient-derived R0R+ CLL TC cells in a chromium- release assay at EC:TC TC ratio of 10: 1 for 6 hours at 37°C with or without anti-CD20 mAb rituximab or anti-ROR mAbs, such as zilovertamab or GE-zilovertamab, at concentrations of 100 ng/ml. Chromium (Cr⁵¹) release was measured after 6-hours of co-culture and percent specific cell lysis was calculated relative to trichloroacetic acid (TCA) treated cells.

[00119] Addition of GE-zilovertamab to co-cultures of EC NK92-CD16v with Cr⁵¹- labeled patient-derived primary R0R+ CLL TC, which expresses both CD20 and R0R1, directed ADCC of CLL targets by NK92-CD16v cells at levels comparable to that directed by rituximab, and such levels were significantly greater than that directed by zilovertamab (P<0.05). No difference was noted for the amount of ADCC directed by GE-zilovertamab, zilovertamab or rituximab using the NK92 effector cells, which were all lower than the respective levels mediated by NK92-CD16v.

[00120] FIG. 7 shows ADCC by NK92-CD16v cells for 17p deleted and p53 mutated R0R1+ CLL cells treated with anti-RORl or anti-CD20 mAb. NK92-CD16v cells were used as EC to effect cytolysis of Cr⁵¹-labeled primary patient-derived 17p deleted and p53 mutated R0R+ CLL TC cells in a chromium-release assay at EC:TC TC ratio of 10: 1 for 6 hours at 37°C with or without anti-CD20 mAb rituximab or anti-ROR mAbs, such as zilovertamab or GE-zilovertamab, at concentrations of 100 ng/ml. Chromium (Cr⁵¹) release was measured after 6-hours of co-culture and percent specific cell lysis was calculated relative to trichloroacetic acid (TCA) treated cells.

[00121] Addition of GE-zilovertamab to co-cultures of EC NK92-C16v with Cr⁵¹-labeled patient-derived primary 17p deleted and p53 mutated R0R+ CLL TC, which expresses both CD20 and R0R1, directed ADCC of CLL target cells at levels comparable to that directed by rituximab, and such levels were significantly greater than that directed by zilovertamab (P<0.05).

[00122] FIG. 8 shows ADCC by peripheral blood mononuclear cells (PBMC) for ROR1+ target cells treated with the GE-zilovertamab anti-RORl mAb. Freshly isolated PBMC were used as EC to effect cytolysis of Cr⁵¹-labeled MEC1 or MEC1-ROR1 target cells (TC) in a chromium-release assay at EC:TC TC ratio of 20: 1 for 6 hours at 37 °C with or without anti- ROR mAbs, zilovertamab or GE-zilovertamab, at concentrations of 100 ng/ml. Chromium (Cr⁵¹) release was measured after 6-hours of co-culture and percent cell lysis was calculated relative to trichloroacetic acid (TCA) treated cells.

[00123] Addition of GE-zilovertamab to co-cultures of PBMC with Cr⁵¹-labeled MEC1 or MEC1-ROR1 target cells mediated ADCC of MEC1-ROR1 targets, but not MEC1 cells that lack ROR1 expression, that was significantly greater than that directed by zilovertamab (P<0.01). For MEC1 target cells that lack ROR1 expression, neither anti-RORl mAb induced levels of ADCC above the level measured using human IgG control antibody.

[00124] FIG. 9 shows ADCC by peripheral blood mononuclear cells (PBMC) for ROR1+ CLL target cells treated with the GE-zilovertamab anti-RORl mAb. Freshly isolated PBMC were used as EC to effect cytolysis of Cr⁵¹-labeled CLL cells in a chromium-release assay at EC:TC TC ratio of 50: 1 for 6 hours at 37°C with or without anti-ROR mAbs, zilovertamab or GE-zilovertamab, at concentrations of 100 ng/ml. Chromium (Cr⁵¹) release was measured after 6-hours of co-culture and percent cell lysis was calculated relative to trichloroacetic acid (TCA) treated cells.

[00125] Addition of GE-zilovertamab to co-cultures of PBMC with Cr⁵¹-labeled CLL target cells mediated ADCC that was significantly greater than that directed by zilovertamab (P<0.05). Additionally, ADCC mediated by GE-zilovertamab could be reduced by addition of anti-human CD 16 mAb to the level mediated by zilovertamab alone (p<0.05).

[00126] Zilovertamab is a first in class antibody for targeting an epitope of ROR1 at the IgG like domain of human ROR1, which is a potential target for treatment ROR1 expressing cancers. However, previously studies have shown minimal ADCC with zilovertamab alone, or only shown modest levels of ADCC in combination with other chemotherapeutic agents (e.g., BCL2 inhibitors) (Rassenti L. et al., Proc. Natl. Acad. Set. USA, 114: 10733, 2017). Previous studies of zilovertamab have failed to achieve levels of ADCC which were comparable to that of other approved antibodies for other targets on leukemic cells (e.g., CD20), and considering that zilovertamab fails to recognize other ROR1 epitopes recognized by competing anti-RORl antibodies, the epitope of ROR1 at the IgG like domain of human R0R1 may be a low density epitope expressed on leukemic cells such that it is a poor target for ADCC activity acting alone. Surprisingly and unexpectedly, afucosylation of zilovertamab significantly increases the ADCC activity of the modified antibody alone to a level which is comparable to that rituximab, an approved antibody which targets a high density epitope at CD20 expressed on leukemic cells. When considering the previous studies in which zilovertamab has failed to achieve appreciable ADCC when acting alone, and the low epitope density of the epitope of R0R1 at the IgG like domain of human R0R1 (e.g., including glutamic acid at position 138), one of skill in the art would not have expected that afucosylation of zilovertamab would significantly increase the ADCC of the modified antibody to a level which is comparable to that of a high density epitope therapeutic, such an anti-CD20 antibody like rituximab.

[00127] Furthermore, ROR1+ CLL cells harboring del(17p) or mutations in TP53 (del(17p)/mTP53), and/or that were resistant to targeted therapies (e.g., inhibitors of BTK or BCL2), were as susceptible to GE-zilovertamab-directed ADCC as were CLL cells without del(17p)/mTP53 from patients who had not had prior therapy.

[00128] These data demonstrate that GE-zilovertamab can direct high-level ADCC lysis of ROR1 -expressing neoplastic cells with greater activity than zilovertamab, encouraging development of clinical studies evaluated GE-zilovertamab for patients with CLL or other ROR1 -positive cancers. Surprisingly and unexpectedly, afucosylation of zilovertamab significantly increases the ADCC activity of the modified antibody alone to a level which is comparable to that rituximab, an approved antibody which targets a high density epitope at CD20 expressed on leukemic cells.

[00129] While preferred embodiments of the present within a composition disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the instant disclosure. It should be understood that various alternatives to the embodiments described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the embodiments disclosed herein and that methods and structures within the scope of these claims and their equivalents be covered thereby. SEQUENCES

Claims

CLAIMS What is claimed is:

1. An R0R1 binding antibody or an R0R1 antigen binding fragment thereof, wherein the R0R1 binding antibody or the R0R1 antigen binding fragment thereof comprises an alteration to increase antibody effector function.

2. The R0R1 binding antibody or the R0R1 antigen binding fragment thereof of claim 1, wherein the alteration to increase antibody effector function increases antibody dependent cell cytotoxicity (ADCC).

3. The R0R1 binding antibody or the R0R1 antigen binding fragment thereof of claim 1 or 2, wherein the alteration to increase antibody effector function comprises one or more amino acid alterations to the Fc region of the antibody compared to a wild type antibody Fc region.

4. The R0R1 binding antibody or the R0R1 antigen binding fragment thereof of claim 3, wherein the one or more amino acid alterations comprise S239D, I332E, A330L, S239D/I332E; S239D/I332E/A330L, or a combination thereof according to EU numbering.

5. The R0R1 binding antibody or the R0R1 antigen binding fragment thereof of claim 1 or 2, wherein the alteration to increase antibody effector function comprises glycoengineering.

6. The R0R1 binding antibody or the R0R1 antigen binding fragment thereof of claim 1 or 2, wherein the alteration to increase antibody effector function comprises an alteration at Asparagine 297 according to EU numbering.

7. The R0R1 binding antibody or the R0R1 antigen binding fragment thereof of claim 1 or 2, wherein the alteration to increase antibody effector function comprises a reduction in fucose.

8. The R0R1 binding antibody or the R0R1 antigen binding fragment thereof of claim 1 or 2, wherein the antibody is afucosylated.

9. The R0R1 binding antibody or the R0R1 antigen binding fragment thereof of claim 1 or 2, wherein the antibody is greater than 95% afucosylated.

10. The ROR1 binding antibody or the ROR1 antigen binding fragment thereof of claim 1 or 2, wherein the antibody is greater than 97% afucosylated.

11. The ROR1 binding antibody or the ROR1 antigen binding fragment thereof of claim 1 or 2, wherein the antibody is greater than 98% afucosylated.

12. The ROR1 binding antibody or the ROR1 antigen binding fragment thereof of claim 1 or 2, wherein the antibody is greater than 99% afucosylated.

13. The ROR1 binding antibody or the ROR1 antigen binding fragment thereof of claim 1 or 2, wherein the antibody is 100% afucosylated.

14. The ROR1 binding antibody or the ROR1 antigen binding fragment thereof of any one of claims 8 to 13, wherein the afucosylated antibody increases antibody dependent cell cytotoxicity (ADCC).

15. The R0R1 binding antibody or the R0R1 antigen binding fragment thereof of any one of claims 1 to 14, wherein the R0R1 binding antibody or the R0R1 antigen binding antibody fragment thereof comprises:

(f) a light chain complementarity determining region 3 (L-CDR3) comprising an amino acid sequence set forth in SEQ ID NO: 6; wherein the anti-RORl antibody or ROR1 -binding antibody fragment thereof binds ROR1.

16. The ROR1 binding antibody or the ROR1 antigen binding fragment thereof of claim 15, wherein the anti-RORl antibody or ROR1 -binding antibody fragment thereof comprises: a heavy chain variable domain comprising at least 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7; and a light chain variable domain comprising at least 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 8.

17. The R0R1 binding antibody or the R0R1 antigen binding fragment thereof of claim 15, wherein the anti-RORl antibody or R0R1 -binding antibody fragment thereof comprises: a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 7; and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8.

18. The R0R1 binding antibody or the R0R1 antigen binding fragment thereof of claim 15, wherein the anti-RORl antibody or R0R1 -binding antibody fragment thereof comprises: a heavy chain comprising at least 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 9; and a light chain comprising at least 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 10.

19. The ROR1 binding antibody or the ROR1 antigen binding fragment thereof of claim 15, wherein the anti-RORl antibody or ROR1 -binding antibody fragment thereof comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 9; and a light chain comprising the amino acid sequence of SEQ ID NO: 10.

20. The ROR1 binding antibody or the ROR1 antigen binding fragment thereof of any one of claims 1 to 19, wherein the anti-RORl antibody or ROR1 -binding antibody fragment thereof comprises an IgGl antibody.

21. The ROR1 binding antibody or the ROR1 antigen binding fragment thereof of any one of claims 1 to 19, wherein the anti-RORl antibody or ROR1 -binding antibody fragment thereof comprises a F(ab), a F(ab’), a F(ab’)2, or an ScFv.

22. The ROR1 binding antibody or the ROR1 antigen binding fragment thereof of any one of claims 1 to 21, wherein the ROR1 binding antibody or the ROR1 antigen binding fragment thereof exhibits antibody dependent cellular cytotoxicity which is greater than or equal to antibody dependent cellular cytotoxicity exhibited by rituximab.

23. The R0R1 binding antibody or the R0R1 antigen binding fragment thereof of claim 22, wherein the antibody dependent cellular cytotoxicity is exhibited against a leukemic cell.

24. The R0R1 binding antibody or the R0R1 antigen binding fragment thereof of claim 23, wherein the leukemic cell comprises a CLL, MZL, or MCL cell.

25. A method of treating a cancer in a subject, comprising administering any of the R0R1 binding antibody or the ROR1 antigen binding fragment thereof of claims 1 to 22 to a subject, thereby treating the cancer in the subject.

26. The method of claim 25, wherein the cancer is an R0R1 expressing cancer.

27. The method of claim 25, wherein the cancer is a leukemia or lymphoma.

28. The method of claim 27, wherein the leukemia or lymphoma is B cell leukemia or lymphoma.

29. The method of claim 27, wherein the leukemia or lymphoma is chronic lymphocytic leukemia (CLL), or small cell lymphocytic leukemia (SLL).

30. The method of claim 27, wherein the leukemia or lymphoma is mantle cell lymphoma (MCL), or marginal zone lymphoma (MZL).

31. The method of claim 25, wherein the cancer is a solid tissue cancer.

32. The method of claim 31, wherein the solid tissue cancer is breast cancer, ovarian cancer, prostate cancer, lung cancer, pancreatic cancer, head and neck cancer, kidney cancer, colon cancer, or stomach cancer.

33. The method of any of claims 25 to 32, wherein the cancer does not comprise: a TP53 mutation, a del(17p) mutation, or one or more mutations in TP53 (del(17p)/mTP53).

34. The method of any of claims 25 to 33, wherein the cancer comprises a TP53 mutation, a del(17p) mutation, or one or more mutations in TP53 (del(17p)/mTP53).

35. The method of claim any of claim 25 to 34, wherein the subject is treatment naive or has not previously been treated with an anticancer agent.

36. The method of claim any of claim 25 to 35, wherein the cancer is resistant to targeted anticancer therapies.

37. The method of claim any of claim 25 to 36, wherein the cancer is resistant to inhibitors of BTK or BCL2.

38. The method of claim 37, wherein the inhibitors of BTK comprises ibrutinib, acalab branebrutinib, elsubrutinib, rutinib, evobrutinib, fenebrutinib, orelabrutinib, rilzabrutinib, tolebrutinib, vecabrutinib, spebrutinib tirabrutinib, nemtabrutinib, AC0058TA, LOXO-305, DTRMWXHS-12, M7583, SN-1011, TG- 1701, or combinations thereof.

39. The method of claim 37, wherein the inhibitors of BCL2 comprise venetoclax, navitoclax, ABT-737, AZD5991, AMG176, AMG397, S64315, ABBV467, or combinations thereof.

40. The method of claim 34, wherein the TP53 mutation is in an exon of the TP53 gene.

41. The method of claim 34, wherein the TP53 mutation the mutation is in an intron of the TP53 gene.

42. The method of claim 34, wherein the TP53 mutation the mutation is a nonsense mutation in the TP53 gene.

43. The method of claim 34, wherein the TP53 mutation the mutation is a missense mutation in the TP53 gene.

44. The method of claim 34, wherein the TP53 mutation the mutation results in an amino acid insertion, deletion, or substitution in a protein encoded by the TP53 gene.

45. The method of claim 25, wherein the cancer is resistant to treatment with a CD20 antibody.

46. The method of claim 25, wherein the cancer is resistant to treatment with rituximab.

47. The method of claim 25, wherein the subject has previously been treated with a chemotherapeutic agent.

48. The method of claim 47, wherein the chemotherapeutic agent comprises a CD20 antibody therapy.

49. The method of claim 47, wherein the chemotherapeutic agent comprises rituximab.

50. The method of claim 47, wherein the chemotherapeutic agent comprises a BCL2 inhibitor.

51. The method of claim 50, wherein the BCL2 inhibitor comprise venetoclax, navitoclax, ABT-737, AZD5991, AMG176, AMG397, S64315, ABBV467, or combinations thereof.

52. The method of claim 47, wherein the subject has previously been treated with a BTK inhibitor.

53. The method of claim 52, wherein the BTK inhibitor comprises ibrutinib, acalab branebrutinib, elsubrutinib, rutinib, evobrutinib, fenebrutinib, orelabrutinib, rilzabrutinib, tolebrutinib, vecabrutinib, spebrutinib, tirabrutinib, nemtabrutinib, AC0058TA, LOXO-305, DTRMWXHS-12, M7583, SN-1011, TG-1701, or combinations thereof.

54. The method of claim 25, wherein administering the R0R1 binding antibody or the R0R1 antigen binding fragment induces antibody dependent cellular cytotoxicity of the cancer.

55. The method of claim 54, wherein the antibody dependent cellular cytotoxicity achieved by the R0R1 binding antibody or the R0R1 antigen binding fragment is greater than or equal to antibody dependent cellular cytotoxicity exhibited by rituximab.

56. The method of claim 55, wherein the R0R1 binding antibody or the R0R1 antigen binding fragment thereof exhibits antibody dependent cellular cytotoxicity which is greater than or equal to antibody dependent cellular cytotoxicity exhibited by rituximab.

57. The method of claim 56, wherein the antibody dependent cellular cytotoxicity is exhibited against a leukemic cell.

58. The method of claim 57, wherein the leukemic cells comprises a CLL, MZL, or MCL cell.