EA042795B1 - HUMANIZED ANTIBODIES AGAINST CD73 - Google Patents

Description

Cross-reference to related applications

This application claims priority to U.S. Provisional Application No. 62/2 65357 filed December 9, 2015, U.S. Provisional Application No. 62/289694 filed February 1, 2016, and U.S. Provisional Application No. 62/346327 filed 6 June 2016, the entire contents of which are hereby provided by reference and for all purposes.

A reference to a sequence list, table, or list-application of a computer program, taken in the form of an ASCII text file

The content of the sequence listing recorded in the file 48517-508001WO_ST25.TXT, created December 9, 2016, 50442 bytes, machine format IBM-PC, MS Windows operating system, is thus incorporated herein by reference.

State of the art

Glycosylphosphatidylinositol-anchored CD73 antigen is believed to be the rate-limiting enzyme in the production of extracellular adenosine (Stagg J, Smyth MJ. Extracellular adenosine triphosphate and adenosine in cancer. Oncogene. 2010; 29:5346-58. doi: 10.1038/onc.2010.292). CD73 can be found constitutively expressed at high levels in various cancer cell types. CD73-derived adenosine is believed to suppress adaptive anti-tumor immune responses, thus stimulating tumor growth and metastasis. There is a need in the art for an anti-CD73 antibody based cancer therapy that inhibits the catalytic activity of CD73 and prevents the ability of circulating tumor cells to invade from vessels and colonize, thus inhibiting metastasis. The present invention addresses these and other needs in the art.

The essence of the invention

In one aspect, the present invention provides a humanized 1E9 antibody comprising a humanized light chain variable region, including a mouse CDR L1, a mouse CDR L2, or a mouse CDR L3, and a humanized heavy chain variable region, including a mouse CDR H1, a mouse CDR H2, or a mouse CDR H3 .

In one aspect, the present invention provides an antibody (eg, a humanized 1E9 antibody). An antibody contains a light chain variable region (eg, a humanized light chain) and a heavy chain variable region (eg, a humanized heavy chain). The variable region of the light chain contains:

(i) L1 CDR (e.g. mouse CDR L1) as set forth in SEQ ID NO: 1, L2 CDR (e.g. mouse CDR L2) as set out in SEQ ID NO: 2, L3 CDR (e.g. mouse CDR L3) as indicated in SEQ ID NO: 3, and (ii) valine at position corresponding to position 2 according to Kabat, methionine at position corresponding to position 4 according to Kabat, aspartic acid or leucine at position corresponding to position 9 according to Kabat, proline or serine at Kabat position 12, lysine or proline at Kabat position 18, alanine at Kabat position 43, proline or serine at Kabat position 60, threonine at position 74 Kabat, asparagine or serine at Kabat position 76, asparagine or serine at Kabat position 77, isoleucine or leucine at Kabat position 78, serine or alanine at Kabat position 80 , glutamine at Kabat position 100, valine at Kabat position 104, glutamic acid or alanine at Kabat position 1, glutamine at Kabat position 3, phenylalanine or threonine at position Kabat position 10, glutamine at Kabat position 11, alanine or leucine at Kabat position 13, threonine at Kabat position 14, valine or proline at Kabat position 15, lysine at Kabat position 16, glutamic acid or aspartic acid at Kabat position 17, threonine at Kabat position 22, lysine at Kabat position 42, arginine at Kabat position Kabat position 45, isoleucine at Kabat position 58, tyrosine at Kabat position 67, phenylalanine at Kabat position 73, tyrosine at Kabat position 85, or phenylalanine at Kabat position position 87 of Kabat.

The heavy chain variable region contains:

(i) CDR H1 (e.g. mouse CDR H1) as set forth in SEQ ID NO: 4, CDRH2 (e.g. mouse CDR H2) as listed in SEQ ID NO: 5, CDR H3 (e.g. mouse CDR H3), as indicated in SEQ ID NO: 6, and (ii) isoleucine at position corresponding to position 37 Kabat, alanine or proline at position corresponding to position 40 Kabat, lysine at position corresponding to position 43 Kabat, serine at position corresponding Kabat position 70, isoleucine or threonine in polo

- 1 042795 Kabat position 75, tryptophan at Kabat position 82, arginine or lysine at Kabat position 83, alanine at Kabat position 84, serine at position 85 Kabat, valine or methionine at Kabat position 89, valine at Kabat position 5, serine at Kabat position 7, valine at Kabat position 11, glutamic acid or lysine at Kabat position 12, isoleucine or valine at Kabat position 20, arginine at Kabat position 38, arginine at Kabat position 66, valine at Kabat position 67, isoleucine at Kabat position 69, alanine at Kabat position 71, lysine at Kabat position 73, threonine at Kabat position 87, glutamic acid at Kabat position 1 , valine at Kabat position 24, arginine at Kabat position 44, methionine at Kabat position 48, leucine at Kabat position 80, or glutamic acid at Kabat position 81 Kabat.

In one aspect, the present invention provides a humanized 1E9 antibody. The 1E9 antibody contains a humanized light chain variable region and a humanized heavy chain variable region. The variable region of the humanized light chain contains:

(i) mouse CDR L1 as listed in SEQ ID NO: 1, mouse CDR L2 as listed in SEQ ID NO: 2, mouse CDR L3 as listed in SEQ ID NO: 3, and (ii) valine at the position, Kabat position 2, methionine at Kabat position 4, aspartic acid or leucine at Kabat position 9, proline or serine at Kabat position 12, lysine or proline at position 18 Kabat, alanine at Kabat position 43, proline or serine at Kabat position 60, threonine at Kabat position 74, asparagine or serine at Kabat position 76, asparagine or serine at Kabat position 77, isoleucine or leucine at Kabat position 78, serine or alanine at Kabat position 80, glutamine at Kabat position 100, valine at Kabat position 104 Kabat, glutamic acid or alanine at Kabat position 1, glutamine at Kabat position 3, phenylalanine or threonine at Kabat position 10, glutamine at Kabat position 11, alanine or leucine at Kabat position 13, threonine at Kabat position 14, valine or proline at Kabat position 15, lysine at Kabat position 16, glutamic acid or aspartic acid at position, Kabat position 17, threonine at Kabat position 22, lysine at Kabat position 42, arginine at Kabat position 45, isoleucine at Kabat position 58, tyrosine at Kabat position 67, phenylalanine at Kabat position 73, tyrosine at Kabat position 85, or phenylalanine at Kabat position 87.

The variable region of a humanized heavy chain contains:

(i) mouse H1 CDR as set out in SEQ ID NO: 4, mouse H2 CDR as set out in SEQ ID NO: 5, and mouse H3 CDR as set out in SEQ ID NO: 6, and (ii) isoleucine at position Kabat position 37, alanine or proline at Kabat position 40, lysine at Kabat position 43, serine at Kabat position 70, isoleucine or threonine at Kabat position 75 , tryptophan at Kabat position 82, arginine or lysine at Kabat position 83, alanine at Kabat position 84, serine at Kabat position 85, valine or methionine at position Kabat position 89, valine at Kabat position 5, serine at Kabat position 7, valine at Kabat position 11, glutamic acid or lysine at Kabat position 12, isoleucine or valine at Kabat position 20, arginine at Kabat position 38, arginine at Kabat position 66, valine at Kabat position 67, isoleucine at Kabat position 69, alanine at position 71 according to Kabat, lysine at position corresponding to position 73 according to Kabat, threonine at position corresponding to position 87 according to Kabat, glutamic acid

- 2 042795 Tu at Kabat position 1, valine at Kabat position 24, arginine at Kabat position 44, methionine at Kabat position 48, leucine at Kabat position 80 according to Kabat or glutamic acid in the position corresponding to position 81 according to Kabat.

In another aspect, the present invention provides an antibody (e.g., a humanized 1E9 antibody) comprising a light chain variable region (e.g., a humanized light chain) and a heavy chain variable region (e.g., a humanized heavy chain), wherein the heavy chain variable region comprises the sequence from SEQ ID NO: 7.

In another aspect, the present invention provides a humanized 1E9 antibody comprising a humanized light chain variable region and a humanized heavy chain variable region, wherein the humanized heavy chain variable region comprises the sequence from SEQ ID NO: 7.

One aspect of the present disclosure relates to an IgG1 antibody (e.g., a humanized IgG1) containing a light chain variable region (e.g., a humanized light chain) and a heavy chain variable region (e.g., a humanized heavy chain), wherein the light chain variable region contains an L1 CDR (e.g. , mouse CDR L1) as listed in SEQ ID NO: 1, CDR L2 (e.g., mouse CDR L2) as listed in SEQ ID NO: 2, CDR L3 (e.g., mouse CDR L3) as listed in SEQ ID NO : 3, and wherein the heavy chain variable region contains an H1 CDR (e.g., mouse CDR H1) as set forth in SEQ ID NO: 4, an H2 CDR (e.g., mouse CDR H2) as set out in SEQ ID NO: 5, CDR H3 (e.g. mouse H3 CDR) as indicated in SEQ ID NO: 6.

One aspect of the present disclosure relates to a humanized IgG1 antibody comprising a humanized light chain variable region and a humanized heavy chain variable region, wherein the humanized light chain variable region comprises a murine CDR L1 as set forth in SEQ ID NO: 1, a murine CDR L2 as set forth in SEQ ID NO: 2, mouse CDR L3 as set forth in SEQ ID NO: 3, and wherein the humanized heavy chain variable region contains mouse CDR H1 as set forth in SEQ ID NO: 4, mouse CDR H2 as set forth in SEQ ID NO : 5, and mouse H3 CDRs as indicated in SEQ ID NO: 6.

One aspect of the present disclosure relates to an IgG4 antibody (e.g., humanized IgG4) comprising a light chain variable region (e.g., a humanized light chain) and a heavy chain variable region (e.g., a humanized heavy chain), wherein the light chain variable region contains an L1 CDR (e.g. , mouse CDR L1) as listed in SEQ ID NO: 1, CDR L2 (e.g., mouse CDR L2) as listed in SEQ ID NO: 2, CDR L3 (e.g., mouse CDR L3) as listed in SEQ ID NO : 3, and wherein the heavy chain variable region contains an H1 CDR (e.g., mouse CDR H1) as set forth in SEQ ID NO: 4, an H2 CDR (e.g., mouse CDR H2) as set out in SEQ ID NO: 5, CDR H3 (e.g. mouse H3 CDR) as indicated in SEQ ID NO: 6.

One aspect of the present disclosure relates to a humanized IgG4 antibody comprising a humanized light chain variable region and a humanized heavy chain variable region, wherein the humanized light chain variable region comprises a mouse CDR L1 as set forth in SEQ ID NO: 1, a mouse CDR L2 as set forth in SEQ ID NO: 2, mouse CDR L3 as set forth in SEQ ID NO: 3, and wherein the humanized heavy chain variable region contains mouse CDR H1 as set forth in SEQ ID NO: 4, mouse CDR H2 as set forth in SEQ ID NO : 5, and mouse H3 CDRs as indicated in SEQ ID NO: 6.

In one aspect, the present invention provides an isolated nucleic acid encoding a 1E9 antibody (eg, a humanized 1E9 antibody) as described herein, including embodiments.

In one aspect, the present invention provides an isolated nucleic acid encoding a humanized 1E9 antibody as described herein, including embodiments.

In another aspect, the present invention provides an isolated nucleic acid encoding an IgG1 antibody (eg, a humanized IgG1 antibody) as described herein, including embodiments.

In another aspect, the present invention provides an isolated nucleic acid encoding a humanized IgG1 antibody as described herein, including embodiments.

In another aspect, the present invention provides an isolated nucleic acid encoding an IgG4 antibody (eg, a humanized IgG4 antibody) as described herein, including embodiments.

In another aspect, the present invention provides an isolated nucleic acid encoding a humanized IgG4 antibody as described herein, including embodiments.

In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an 1E9 antibody (eg, a humanized 1E9 antibody) as described herein, including embodiments thereof, and a pharmaceutically acceptable excipient.

- 3 042795

In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the humanized 1E9 antibody described herein, including embodiments thereof, and a pharmaceutically acceptable excipient.

In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an IgG1 antibody (eg, a humanized IgG1 antibody) as described herein, including embodiments thereof, and a pharmaceutically acceptable excipient.

In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a humanized IgG1 antibody as described herein, including embodiments thereof, and a pharmaceutically acceptable excipient.

In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an IgG4 antibody (eg, a humanized IgG4 antibody) as described herein, including embodiments thereof, and a pharmaceutically acceptable excipient.

In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a humanized IgG4 antibody as described herein, including embodiments thereof, and a pharmaceutically acceptable excipient.

In one aspect, the present invention relates to a method of treating cancer in a subject in need thereof. The method includes administering to a subject a therapeutically effective amount of an 1E9 antibody (eg, a humanized 1E9 antibody) provided herein, including embodiments thereof, thereby treating the subject's cancer.

In one aspect, the present invention relates to a method of treating cancer in a subject in need thereof. The method includes administering to a subject a therapeutically effective amount of a humanized 1E9 antibody provided herein, including embodiments thereof, thereby treating a cancer in the subject.

In one aspect, the present invention relates to a method of treating cancer in a subject in need thereof. The method includes administering to a subject a therapeutically effective amount of an IgG1 antibody (eg, a humanized IgG1 antibody) provided herein, including embodiments thereof, thereby treating the subject's cancer.

In one aspect, the present invention relates to a method of treating cancer in a subject in need thereof. The method includes administering to a subject a therapeutically effective amount of a humanized IgG1 antibody provided herein, including embodiments thereof, thereby treating a cancer in the subject.

In one aspect, the present invention relates to a method of treating cancer in a subject in need thereof. The method includes administering to a subject a therapeutically effective amount of an IgG4 antibody (eg, a humanized IgG4 antibody) provided herein, including embodiments thereof, thereby treating the subject's cancer.

In one aspect, the present invention relates to a method of treating cancer in a subject in need thereof. The method includes administering to a subject a therapeutically effective amount of a humanized IgG4 antibody provided herein, including embodiments thereof, thereby treating the subject's cancer.

In one aspect, the present invention relates to a method for inhibiting cell proliferation. The method includes:

(i) contacting a cell with an IgG1 antibody (eg, a humanized IgG1 antibody) as described herein, including embodiments thereof, thereby obtaining a contacted cell;

(ii) an IgG1 antibody (eg, a humanized IgG1 antibody) is allowed to bind the CD73 antigen on the contacted cell, thereby inhibiting cell proliferation.

In one aspect, the present invention relates to a method for inhibiting cell proliferation. The method includes:

(i) contacting a cell with a humanized IgG1 antibody as described herein, including embodiments thereof, thereby obtaining a contacted cell;

(ii) the humanized IgG1 antibody is allowed to bind the CD73 antigen on the contacted cell, thereby inhibiting cell proliferation.

In one aspect, the present invention relates to a method for inhibiting cell proliferation. The method includes:

(i) contacting the cell with an IgG4 antibody (e.g., a humanized IgG4 antibody) as described herein, including embodiments, thereby obtaining

- 4 042795 niye brought into contact cells;

(ii) an IgG4 antibody (eg, a humanized IgG4 antibody) is allowed to bind the CD73 antigen on the contacted cell, thereby inhibiting cell proliferation.

In one aspect, the present invention relates to a method for inhibiting cell proliferation. The method includes:

(i) contacting a cell with a humanized IgG4 antibody as described herein, including embodiments thereof, thereby obtaining a contacted cell;

(ii) the humanized IgG4 antibody is allowed to bind the CD73 antigen on the contacted cell, thereby inhibiting cell proliferation.

In another aspect, the present invention provides an anti-CD73 antibody. An anti-CD73 antibody binds the same epitope as an 1E9 antibody, wherein the 1E9 antibody contains a light chain variable region (e.g., a humanized light chain variable region), including CDR L1 (e.g., mouse CDR L1), CDR L2 (e.g., CDR L2 mouse) or L3 CDR (e.g., mouse CDR L3), and a heavy chain variable region (e.g., humanized heavy chain variable region), including CDR H1 (e.g., mouse CDR H1), CDR H2 (e.g., mouse CDR H2), or CDR H3 (e.g. mouse CDR H3).

In another aspect, the present invention provides an anti-CD73 antibody. The anti-CD73 antibody binds the same epitope as the 1E9 antibody, wherein the 1E9 antibody contains a humanized light chain variable region, including mouse CDR L1, mouse CDR L2, or mouse CDR L3, and a humanized heavy chain variable region, including mouse CDR H1, CDR H2 mouse or CDR H3 mouse.

In another aspect, the present invention provides an anti-CD73 antibody. The CD73 antibody binds the same epitope as the 1E9 antibody, wherein the 1E9 antibody contains a light chain variable region (eg, a humanized light chain variable region) and a heavy chain variable region (eg, a humanized heavy chain variable region). The variable region of the light chain contains:

(i) L1 CDR (e.g. mouse CDR L1) as set forth in SEQ ID NO: 1, L2 CDR (e.g. mouse CDR L2) as set out in SEQ ID NO: 2, L3 CDR (e.g. mouse CDR L3) as indicated in SEQ ID NO: 3, and (ii) valine at position corresponding to position 2 according to Kabat, methionine at position corresponding to position 4 according to Kabat, aspartic acid or leucine at position corresponding to position 9 according to Kabat, proline or serine at Kabat position 12, lysine or proline at Kabat position 18, alanine at Kabat position 43, proline or serine at Kabat position 60, threonine at position 74 Kabat, asparagine or serine at Kabat position 76, asparagine or serine at Kabat position 77, isoleucine or leucine at Kabat position 78, serine or alanine at Kabat position 80 , glutamine at Kabat position 100, valine at Kabat position 104, glutamic acid or alanine at Kabat position 1, glutamine at Kabat position 3, phenylalanine or threonine at position Kabat position 10, glutamine at Kabat position 11, alanine or leucine at Kabat position 13, threonine at Kabat position 14, valine or proline at Kabat position 15, lysine at Kabat position 16, glutamic acid or aspartic acid at Kabat position 17, threonine at Kabat position 22, lysine at Kabat position 42, arginine at Kabat position 4 5 Kabat, isoleucine at Kabat position 58, tyrosine at Kabat position 67, phenylalanine at Kabat position 73, tyrosine at Kabat position 85, or phenylalanine at compliant with regulation 87 of Kabat.

The variable region of a humanized heavy chain contains:

(i) CDR H1 (e.g. mouse CDR H1) as set forth in SEQ ID NO: 4, CDR H2 (e.g. mouse CDR H2) as listed in SEQ ID NO: 5, CDR H3 (e.g. mouse CDR H3) as indicated in SEQ ID NO: 6, and (ii) isoleucine at position corresponding to position 37 Kabat, alanine or proline at position corresponding to position 40 Kabat, lysine at position corresponding to position 43 Kabat, serine at position, Kabat position 70, isoleucine or threonine at Kabat position 75, tryptophan at Kabat position 82, arginine or lysine at Kabat position 83, alanine at Kabat position 84, serine at Kabat position 85, valine or methionine at Kabat position 89, valine at

- 5 042795 Kabat position 5, serine at Kabat position 7, valine at Kabat position 11, glutamic acid or lysine at Kabat position 12, isoleucine or valine at position, Kabat position 20, arginine at Kabat position 38, arginine at Kabat position 66, valine at Kabat position 67, isoleucine at Kabat position 69, alanine at Kabat position 71, lysine at Kabat position 73, threonine at Kabat position 87, glutamic acid at Kabat position 1, valine at Kabat position 24, arginine at position , corresponding to position 44 according to Kabat, methionine in position corresponding to position 4 8 according to Kabat, leucine in position corresponding to position 80 according to Kabat, or glutamic acid in position corresponding to position 81 according to Kabat.

In another aspect, the present invention provides an anti-CD73 antibody. The CD73 antibody binds the same epitope as the 1E9 antibody, wherein the 1E9 antibody contains a humanized light chain variable region and a humanized heavy chain variable region. The variable region of the humanized light chain contains:

(i) mouse CDR L1 as listed in SEQ ID NO: 1, mouse CDR L2 as listed in SEQ ID NO: 2, mouse CDR L3 as listed in SEQ ID NO: 3, and (ii) valine at the position, Kabat position 2, methionine at Kabat position 4, aspartic acid or leucine at Kabat position 9, proline or serine at Kabat position 12, lysine or proline at position 18 Kabat, alanine at Kabat position 43, proline or serine at Kabat position 60, threonine at Kabat position 74, asparagine or serine at Kabat position 76, asparagine or serine at Kabat position 77, isoleucine or leucine at Kabat position 78, serine or alanine at Kabat position 80, glutamine at Kabat position 100, valine at Kabat position 104 Kabat, glutamic acid or alanine at Kabat position 1, glutamine at Kabat position 3, phenylalanine or threonine at Kabat position 10, glutamine at Kabat position 11, alanine or leucine at Kabat position 13, threonine at Kabat position 14, valine or proline at Kabat position 15, lysine at Kabat position 16, glutamic acid or aspartic acid at position, Kabat position 17, threonine at Kabat position 22, lysine at Kabat position 42, arginine at Kabat position 45, isoleucine at Kabat position 58, tyrosine at Kabat position 67, phenylalanine at Kabat position 73, tyrosine at Kabat position 85, or phenylalanine at Kabat position 87.

The variable region of a humanized heavy chain contains:

(i) mouse H1 CDR as set forth in SEQ ID NO: 4, mouse H2 CDR as set out in SEQ ID NO: 5, and mouse H3 CDR as set out in SEQ ID NO: 6, and (ii) isoleucine at position Kabat position 37, alanine or proline at Kabat position 40, lysine at Kabat position 43, serine at Kabat position 70, isoleucine or threonine at Kabat position 75 , tryptophan at Kabat position 82, arginine or lysine at Kabat position 83, alanine at Kabat position 84, serine at Kabat position 85, valine or methionine at position Kabat position 89, valine at Kabat position 5, serine at Kabat position 7, valine at Kabat position 11, glutamic acid or lysine at Kabat position 12, isoleucine or valine at Kabat position 20, arginine at Kabat position 38, arginine at Kabat position 66, valine at Kabat position 67, isoleucine at Kabat position 69, alanine at Kabat position 71, lysine at Kabat position 73, threonine at Kabat position 87, glutamic acid at Kabat position 1, valine at Kabat position 24 , arginine at Kabat position 44, methionine at Kabat position 48, leucine at position 80

- 6 042795 according to Kabat, or glutamic acid in the position corresponding to position 81 according to Kabat.

Brief description of the figures

Fig. 1: Human T cells isolated by negative selection cultured with the indicated treatment for 5 days. Cell proliferation is analyzed by dilution of the tracking dye Cell Trace Violet.

Fig. 2: Human T cells isolated by negative selection cultured with the indicated treatment for 5 days. CD25 expression was analyzed by flow cytometry.

Fig. 3A-3E: MDA-MB-231 cells, human breast cell line CD73, were incubated with the indicated humanized antibodies (CPX-002 in Figure 3A; CPX-005 in Figure 3B; CPX-006 in Figure 3C; CPX- 004 in Fig. 3D; CPX-003 in Fig. 3E) over a range of concentrations for 1 hour at 37°C. Cells were washed in phosphate-free buffer and then incubated with 250 μM AMP for 20 min at 37°C. Conditioned media was collected from each well and phosphate levels were determined using a malachite green detection kit (Sensolyte MG phosphate kit, AnaSPEC). Absolute phosphate levels were determined by interpolation to a standard curve. The analysis was carried out under conditions of 3 different pH: 7.2, 6.7 or 6.3. Cells were equilibrated in an appropriate pH buffer by washing before adding the antibody. The rest of the analysis was carried out in a buffer of the specified pH.

Fig. 4A and FIG. 4V:

Fig. 4A: Heavy chain variable domain alignment

The variable domains of the 10 best responding clones were aligned with the original clone BAP09401 (LC of chimeric 1E9 (SEQ ID NO: 28)). Amino acid residues identical to the original clone are represented by dots, only amino acid differences are shown. Four unique HC variable domains were identified among the 10 clones with the best response. Sequences are listed (top to bottom) based on the identity of framework 3, framework 2, and framework 1. CDRs are boxed. The sequence indicated for BAP094-hum01-HC corresponds to SEQ ID NO: 42; the sequence indicated for BAP094-hum02-HC corresponds to SEQ ID NO: 43; the sequence indicated for BAP094-hum06-HC corresponds to SEQ ID NO: 47; the sequence indicated for BAP094-hum07-HC corresponds to SEQ ID NO: 48; the sequence indicated for BAP094-hum08-HC corresponds to SEQ ID NO: 49; the sequence indicated for BAP094-hum03HC corresponds to SEQ ID NO: 44; the sequence indicated for BAP094-hum09-HC corresponds to SEQ ID NO: 50; the sequence indicated for BAP094-hum04-HC corresponds to SEQ ID NO: 45; the sequence indicated for BAP094-hum05-HC corresponds to SEQ ID NO: 46; the sequence given for BAP094-huml0-HC corresponds to SEQ ID NO: 51.

Fig. 4B: Light chain variable domain alignment

The variable domains of the clones with the best response were aligned with the original clone BAP094-01 (LHC chimeric 1E9 (SEQ ID NO: 29)). Amino acid residues identical to the original clone are represented by dots, only amino acid differences are shown. 5 unique LC variable domains were identified among the 10 clones with the best response. Sequences are listed (top to bottom) based on the identity of framework 3, framework 2, and framework 1. CDRs are boxed. The sequence indicated for BAP094-hum02-LC corresponds to SEQ ID NO: 32; the sequence indicated for BAP094-hum03-LC corresponds to SEQ ID NO: 33; the sequence indicated for BAP094-hum04-LC corresponds to SEQ ID NO: 34; the sequence indicated for BAP094-hum01-LC corresponds to SEQ ID NO: 31; the sequence indicated for BAP094hum07-LC corresponds to SEQ ID NO: 37; the sequence indicated for BAP094-hum08-LC corresponds to SEQ ID NO: 38; the sequence indicated for BAP094-hum09-LC corresponds to SEQ ID NO: 39; the sequence indicated for BAP094-huml0-LC corresponds to SEQ ID NO: 40; the sequence indicated for BAP094-hum05-LC corresponds to SEQ ID NO: 35; the sequence given for BAP094-hum06-LC corresponds to SEQ ID NO: 36.

Fig. 5: Cell-based ELISA for selected 10 best responding humanized variants

CHO cells were seeded into 6-well plates, transfected with BAP094-01 (i.e., a 1E9 chimeric antibody containing the variable light chain from SEQ ID NO: 30 and the variable heavy chain from SEQ ID NO: 41), selected humanized variants, or vector only, and cultured at 37° C. in DMEM with 10% fetal bovine serum. Supernatants were collected 48 hours after transfection. The concentration of antibodies in the supernatant was determined by a quantitative ELISA method, where unknown values were interpolated to a standard curve. Supernatants were diluted to 50 ng/ml in growth medium (DMEM with 10% serum). 100 µl of the diluted supernatant was added to the wells of a 96-well plate containing MDA-MB231 cells (3 x 10 ⁴ MDAMB231 cells/well plated the day before) and incubated at room temperature for 1 hour. Anti-human IgG (H+L), conjugated to HRP (Promega #W4031) was used as a secondary detection antibody. Reactions were stopped 6 min after the addition of TMB

- 7 042795 into the wells and read immediately.

Fig. 6: CPX-006 attenuates AMP-dependent suppression of IFN-gamma. Human peripheral blood mononuclear cells were incubated with 1 μg/ml of anti-CD3 and anti-CD28 antibodies to stimulate T cell proliferation. 1 mM AMP was added to cultures as a substrate for cellular CD73 to be converted to the immunosuppressive adenosine. Cultures were also incubated with anti-CD73 antibody (CPX-006) or isotype control over a range of concentrations. After 4 days in culture, levels of interferon-gamma (IFN-gamma) were measured in the medium after cell culture by AlphaLISA. The fluorescence signal is proportional to the amounts of IFN-gamma present. CPX-006 attenuated AMP-dependent suppression of T cell activity as measured by IFN-gamma secretion.

Fig. 7: CPX-006 attenuates AMP-dependent suppression of T cell proliferation. Human peripheral blood mononuclear cells were labeled with Cell Trace Violet and incubated with 1 μg/ml of anti-CD3 antibody and anti-CD28 antibody to stimulate T cell proliferation. 1 mM AMP was added to cultures as a substrate for cellular CD73 to be converted to the immunosuppressive adenosine. Cultures were also incubated with anti-CD73 antibody (CPX-006) or isotype control over a range of concentrations. After 4 days in culture, cells were stained with fluorophore-labeled anti-CD3 antibody and analyzed by flow cytometry. Proliferation of CD3+ T cells were selected as events diluted with Cell Trace Violet dye. CPX-006 attenuated AMP-dependent suppression of T-cell proliferation.

Detailed description

Definitions

While various embodiments and aspects of the present invention are shown and described herein, those skilled in the art will recognize that such embodiments and aspects are by way of example only. Numerous variations, modifications and substitutions will now become apparent to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used in the practice of the invention.

The section headings used in this specification are for organizational purposes only and should not be construed as limiting the described subject matter. The entire contents of all documents, or parts of documents, cited in the application, including, without limitation, patents, patent applications, articles, books, manuals, and monographs, are hereby expressly incorporated by reference for all purposes.

Antibodies are large, complex molecules (molecular weight ~150,000 or approximately 1320 amino acids) with a complex internal structure. The natural antibody molecule contains two identical pairs of polypeptide chains, where each pair has one light chain and one heavy chain. Each light chain and heavy chain in turn consists of two regions: a variable (V) region involved in target antigen binding and a constant (C) region interacting with other components of the immune system. The light and heavy chain variable regions converge in 3-dimensional space to form a variable region that binds an antigen (eg, a cell surface receptor). Within each light or heavy chain variable region, there are three short fragments (averaging 10 amino acids in length) called complementarity determining regions (CDRs). The six CDRs in the antibody variable domain (three from the light chain and three from the heavy chain) are folded together in 3D space to form the actual antibody binding site that mates with the target antigen. The position and length of the CDR is precisely defined in Kabat, E. et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1983, 1987. The part of the variable region not contained in the CDR is called the framework region (FR), which forms the environment for the CDR.

The terms CDR L1, CDR L2, and CDR L3, as used herein, refer to complementarity determining regions (CDRs) 1, 2, and 3 from an antibody variable light (L) chain. Similarly, the terms CDR H1, CDR H2, and CDR H3, as used herein, refer to complementarity determining regions (CDRs) 1, 2, and 3 of an antibody variable heavy (H) chain.

The term antibody is used in accordance with its well-known meaning in this field. Antibodies exist, for example, in the form of intact immunoglobulins or in the form of a number of well-characterized fragments obtained by digestion using various peptidases. Thus, for example, pepsin cleaves an antibody below the hinge disulfide bonds to produce F(ab)' ₂ , a Fab dimer that is itself a light chain linked to V _H -C _H1 via a disulfide bond. F(ab)' ₂ can be reduced under mild conditions to break the disulfide bond in the hinge region, thus converting the F(ab)'2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab with a portion of the hinge region (see Fundamental Immunology (Paul ed., ^3d ed. 1993). While different antibody fragments are defined in terms of intact antibody cleavage, one of skill in the art will appreciate that such fragments can be synthesized

- 8 042795 de novo, either chemically or using the recombinant DNA method. Thus, the term antibody, as used herein, also includes antibody fragments either generated by modification of full-length antibodies, or synthesized de novo using recombinant DNA techniques (e.g., single-stranded Fv), or identified using phage display libraries (see , for example, McCafferty et al., Nature 348: 552-554 (1990)).

Any method known in the art can be used to generate monoclonal or polyclonal antibodies (see, for example, Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy (1985)). Monoclonal antibodies (mAbs) refer to antibodies derived from a single clone. Methods for producing single chain antibodies (US Patent No. 4946778) can be adapted to obtain antibodies against the polypeptides of this invention. Also, transgenic mice or other organisms such as other mammals can be used to express humanized antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, for example, McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10: 779-783 (1992)).

The mAb epitope is the region on the antigen to which the mAb binds. Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) the binding of the other to the antigen. That is, a 1x, 5x, 10x, 20x, or 100x excess of one antibody inhibits the binding of the other by at least 30%, but preferably by 50, 75, 90, or even 99%, as measured by competitive binding assay (see, for example, Junghans et al., Cancer Res. 50:1495, 1990). Alternatively, two antibodies have the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other. Two antibodies have overlapping epitopes if some amino acid mutation that reduces or eliminates the binding of one antibody reduces or eliminates the binding of the other.

A ligand refers to an agent, such as a polypeptide or other molecule, capable of binding to a receptor.

A label or detectable group is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, labels that can be used include 32P, fluorescent dyes, electron dense reagents, enzymes (eg, as commonly used in ELISA), biotin, digoxigenin or haptens, and proteins, or other substances that can be made detectable, for example, by incorporating a radioactive label into a peptide or antibody specifically reactive with the target peptide. Any suitable method known in the art can be used to conjugate an antibody to a label, for example using the methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.

Bringing into contact is used in accordance with its simple conventional meaning, and it refers to the process of allowing at least two separate molecules (for example, chemical compounds, including biomolecules or cells) to become close enough for a reaction, interaction or physical contact. It should be understood, however, that the resulting reaction product may be formed directly from the reaction between the added reactants or from an intermediate from one or more of the added reactants that may be formed in the reaction mixture.

The term bringing into contact may include allowing two substances to react, interact or physically contact, where the two substances may be, for example, a biotin domain as described herein and a biotin binding domain. In some embodiments, contacting includes, for example, allowing a humanized antibody, as described herein, to interact with the CD73 antigen.

The terms polypeptide, peptide, and protein are used interchangeably herein to refer to a polymer of amino acid residues, where the polymer may, in some embodiments, be conjugated to a group that is not composed of amino acids. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and to non-natural amino acid polymers. A fusion protein refers to a chimeric protein encoding two or more separate protein sequences recombinantly expressed as a single moiety.

The term peptidyl and peptidyl group denote a monovalent peptide.

The term amino acid refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics, which function in a similar manner to naturally occurring amino acids. Natural amino acids are amino acids encoded genetically

- 9 042795, as well as later modified amino acids, such as hydroxyproline, γ-carboxyglutamate and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as the naturally occurring amino acid, i.e., an α-carbon atom bonded to hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium . Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as the naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a similar way to naturally occurring amino acids. The terms non-natural amino acid and natural amino acid refer to amino acid analogs, synthetic amino acids, and amino acid mimetics not found in nature.

Amino acids may be referred to herein using the commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be designated by conventional single-letter codes.

Conservatively modified variants are applicable to both amino acid sequences and nucleic acid sequences. With respect to specific nucleic acid sequences, conservatively modified variants refer to nucleic acids encoding identical or substantially identical amino acid sequences. Due to the degeneracy of the genetic code, multiple nucleic acid sequences can code for any given protein. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Thus, at each position where alanine is defined by a codon, the codon can be changed to any of the corresponding codons described, without changing the encoded polypeptide. Such nucleic acid variants are silent variants, which are one kind of conservatively modified variants. Each nucleic acid sequence herein that encodes a polypeptide also describes each possible silent nucleic acid variant. One skilled in the art will recognize that every codon in a nucleic acid (with the exception of AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to produce a functionally identical molecule. Accordingly, each silent nucleic acid variant that encodes a polypeptide is meant in each described sequence.

With regard to amino acid sequences, one skilled in the art will recognize that individual substitutions, deletions, or additions in a nucleic acid, peptide, polypeptide, or protein sequence that alter, add, or delete one amino acid or a small percentage of amino acids in the encoded sequence constitute a conservatively modified variant. when the change results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables representing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, the polymorphic variants, interspecies homologues and alleles of the invention.

Each of the following eight groups contains amino acids that are conservative substitutions for each other:

1) alaniya (A), glycine (G);

2) aspartic acid (D), glutamic acid (E);

3) asparagine (N), glutamine (Q);

4) arginine (R), lysine (K);

5) isoleucine (I), leucine (L), methionine (M), valine (V);

6) phenylalanine (F), tyrosine (Y), tryptophan (W);

7) serine (S), threonine (T) and

8) cysteine (C), methionine (M) (see, for example, Creighton, Proteins (1984)).

Percent sequence identity is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (which contains no additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which an identical nucleic acid base or identical amino acid residue occurs in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain percent sequence identity.

The terms identical or percent identical, in the context of two or more consecutive

- 10 042795 nucleic acid or polypeptide stems refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65, 70, 75, 80%, 85%, 90%, 95%, 98%, or 99% identity over a specified region, e.g., the entire polypeptide sequence of the invention, or individual domains of the polypeptides of the invention), when compared and aligned for maximum match over the comparison window or specified region, as measured using one of the following sequence comparison algorithms or through manual alignment and visual inspection. Such sequences are then referred to as substantially identical. This definition also applies to a sequence that is complementary to the test sequence. Optionally, the identity is present over a region having a length of at least about 50 nucleotides, or more preferably over a region having a length of 100-500 or 1000 or more nucleotides. The present invention relates to polypeptides substantially identical to any of SEQ ID NOs: 30-51.

For sequence comparisons, typically one sequence acts as a reference sequence against which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are assigned, if necessary, and algorithm program parameters are assigned to the sequences. You can use the default program settings, or you can assign alternative settings. The sequence comparison algorithm then calculates the percent sequence identity for the test sequences relative to the reference sequence, based on the program parameters.

A comparison window, as used herein, includes a reference to a fragment from any one of the numbers of contiguous positions selected from the group consisting of, for example, the full length sequence, or from 20 to 600, from about 50 to about 200, or from about 100 to about 150 amino acids or nucleotides, in which the sequence can be compared with a reference sequence of the same number of contiguous positions after optimal alignment of the two sequences. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, via the Needleman and Wunsch (1970) homology region alignment algorithm J. Mol. Biol. 48:443, by means of Pearson and Lipman's (1988) similarity search method Proc. Nat'l. Acad. sci. USA 85:2444, through computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Dr., Madison, WI), or through manual alignment and visual inspection (see, for example, Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

One example of an algorithm suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410 respectively. The BLAST analysis software is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying sequence pairs with a high score of similarity (HSP) by identifying short words of length W in the query sequence that either match or meet some positive threshold T when aligned with a word of the same length in the database sequence. T denotes a threshold similarity score for adjacent words (Altschul et al., supra). These initial adjacent word hits act as seeds to initiate searches to find longer HSPs containing them. The word hits expand in both directions along each sequence as long as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, the score matrix is used to calculate the cumulative score. Word hit expansion in each direction is terminated when the cumulative alignment score is reduced by the X value from its maximum achieved value; the cumulative score tends to zero or below due to the accumulation of one or more negatively evaluated residual alignments; or the end of any of the sequences has been reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses the default word length (W) 11, expectation (E) 10, M=5, N=-4, and comparison of both strands. For amino acid sequences, the BLASTP program uses a default word length of 3 and an expectation (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915), alignments (B ) 50, waiting (E) 10,

- 11 042795

M=5, N=-4 and comparison of both chains.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum of probabilities (P(N)), which indicates the probability that two nucleotide or amino acid sequences can match by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum of probabilities when comparing the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or two polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid has immunological cross-reactivity with antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. . Thus, the polypeptide is typically substantially identical to the second polypeptide, for example, when the two peptides differ only in conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules, or their complementary molecules, hybridize to each other under stringent conditions, as described below. Another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.

An amino acid residue in an antibody corresponds to a given residue when it occupies the same important structural position within the antibody as the given residue. For example, a selected residue in a comparison antibody corresponds to position 48 (according to the Kabat numbering system as described herein) in an antibody provided herein when the selected residue has the same significant relationship with Kabat position 48 as judged by using methods applicable in the art. For example, a comparator antibody can be aligned for maximum sequence homology to an antibody provided herein, and a position in the aligned comparator that aligns with Kabat position 48 can be determined to match it. Alternatively, instead of (or in addition to) the primary sequence alignment as described above, a three-dimensional structural alignment can also be used, e.g., where the structure of the antibody being compared is aligned to closely match the antibody described herein and the overall structures are compared. In this case, the amino acid that occupies the same important position as Kabat position 48 in the structural model can be called the corresponding one.

The term isolated in relation to a protein means that the protein is essentially free from other cellular components with which it is associated in its natural state. It is preferably in a homogeneous state, although it may either be dry or in aqueous solution. Purity and homogeneity are typically determined using analytical chemistry methods such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The protein, which is the predominant molecule in the formulation, is essentially purified. The term "purified" means that substantially one band is obtained for the protein in the electrophoretic gel. In particular, this means that the protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.

The phrase specifically (or selectively) binds to an antibody or is specifically (or selectively) immunoreactive, in relation to a protein or peptide, refers to a binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other biological substances. Thus, under the indicated immunoassay conditions, said antibodies bind to a particular protein at least twice as much as background binding, and do not substantially bind in significant amounts to other proteins present in the sample. Typically, a specific or selective response will result in a signal at least twice the background or noise signal, and more specifically, more than 10 to 100 times the background.

A cell, as used herein, refers to a cell that performs a metabolic or other function sufficient to maintain or replicate its genomic DNA. The cell can be identified by methods well known in the art, including, for example, the presence of an intact membrane, staining with a particular dye, the ability to procreate or, in the case of a gamete, the ability to combine with a second gamete to produce viable progeny. The cells may include prokaryotic and eukaryotic cells. Prokaryotic cells include, but are not limited to, bacteria. Eukaryotic cells include, but are not limited to, yeast cells and cells derived from plants and animals, such as mammalian, insect (eg spodoptera) and human cells.

- 12 042795

As defined herein, the term inhibition, inhibit, inhibitory, and the like. in relation to a protein-inhibitor interaction (e.g. humanized 1E9CD73 antibody) means a negative effect (e.g. decreasing) on the activity or function of a protein (e.g. a decrease in the catalytic activity of CD73) compared to the activity or function of the protein in the absence of an inhibitor (e.g. a humanized 1E9 antibody). ). In some embodiments, inhibition refers to a reduction in a disease or symptoms of a disease. Thus, inhibition includes, at least in part, in part, or completely, blocking stimulation, reducing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signaling, or enzymatic activity, or protein amount. Similarly, an inhibitor is a compound or protein that inhibits CD73 activity, for example, by binding to, partially or completely blocking, reducing, preventing, delaying, inactivating, desensitizing, or down-regulating an enzymatic activity (eg, CD73 catalytic activity).

The agents of the invention are often administered in the form of pharmaceutical compositions containing the active drug and a variety of other pharmaceutically acceptable ingredients (See Remington's Pharmaceutical Science ( ^15th ed., Mack Publishing Company, Easton, Pennsylvania, 1980)).

The preferred form depends on the intended route of administration and therapeutic application. The compositions may also include, depending on the desired composition, pharmaceutically acceptable, non-toxic carriers or diluents, defined as conventional binders for formulating pharmaceutical compositions for administration to animals or humans. The diluent is chosen so that it does not affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution and Hank's solution. In addition, the pharmaceutical composition or composition may also include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, and the like.

The compositions can be administered for therapeutic or prophylactic treatment. In therapeutic applications, the compositions are administered to a patient suffering from a disease (eg, cancer) at a therapeutically effective dose. Amounts effective for this application may depend on the severity of the disease and the general health of the patient. Single or multiple administrations of the compositions may be administered depending on the dose and frequency, as needed and as tolerated by the patient. The patient or subject for the purposes of the present invention includes both humans and other animals, in particular mammals. Thus, the methods can be used for both human therapy and veterinary applications. In a preferred embodiment, the patient is a mammal, preferably a primate, and in a most preferred embodiment, the patient is a human.

Formulations suitable for oral administration may consist of:

(a) liquid formulations such as an effective amount of packaged nucleic acid suspended in diluents such as water, saline or PEG 400;

(b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, in the form of liquids, solids, granules or gelatin;

(c) suspensions in a suitable liquid; and (d) suitable emulsions.

Tablet forms may include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid and other excipients, colors, fillers, binders agents, diluents, buffering agents, wetting agents, preservatives, flavoring agents, colorants, disintegrants and pharmaceutically compatible carriers. Lozenge forms may contain the active ingredient in a flavoring agent such as sucrose, as well as lozenges containing the active ingredient in an inert base such as gelatin and glycerin or sucrose and gum arabic emulsions, gels and the like containing, in addition to active ingredient, carriers known in the art.

Pharmaceutical compositions may also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids, and copolymers (such as latex-functionalized sepharose (TM), agarose, cellulose, etc.), polymeric amino acids, amino acid copolymers, and liquid aggregates (such as oil droplets or liposomes). In addition, these carriers can function as immunostimulatory agents (ie, adjuvants).

The compositions provided herein, alone or in combination with other suitable ingredients, can be prepared in the form of aerosol formulations (i.e., they can be sprayed) for administration via inhalation. Aerosol formulations can be placed in suitable pressurized propellants such as dichlorodifluoromethane, propane, nitrogen, and the like.

Suitable formulations for rectal administration include, for example, suppositories consisting of

- 13 042795 packaged nucleic acid base for suppositories. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. In addition, gelatin rectal capsules consisting of a combination of the selected compound with a base including, for example, liquid triglycerides, polyethylene glycols, and paraffinic hydrocarbons can also be used.

Formulations suitable for parenteral administration, for example, via such routes as intra-articular (into the joints), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal and subcutaneous, include aqueous and non-aqueous, isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostatics and solutes to make the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickeners, stabilizers and preservatives. In the practice of this invention, the compositions may be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically, or intrathecally. Parenteral administration, oral administration, and intravenous administration are the preferred routes of administration. Compound formulations can be provided in hermetically sealed single dose or multiple dose containers such as ampoules and vials.

Injectable solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described. Cells transduced with nucleic acids for ex vivo therapy can also be administered intravenously or parenterally as described above.

The pharmaceutical composition is preferably in unit dosage form. In this form, the preparation is subdivided into unit doses containing appropriate amounts of the active ingredient. The unit dosage form may be a packaged preparation, where the package contains discrete amounts of the drug, such as packaged tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form may be a capsule, tablet, sachet, or lozenge itself, or may be a suitable amount of any of these packaged forms. The composition may, if desired, also contain other compatible drugs.

Combined administration means co-administration, using separate formulations or a single pharmaceutical formulation, and sequential administration in any order, where preferably there is a period of time when both (or all) active substances simultaneously exhibit their biological activity.

Effective dosages of the compositions provided herein will vary depending on many different factors, including the routes of administration, the target site, the physiological state of the patient, whether the patient is a human or an animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. However, one skilled in the art can immediately determine suitable and/or equivalent doses by consulting the doses of approved compositions for the treatment and prevention of cancer for guidance.

The terms disease or condition refer to the condition or general health of a patient or subject that can be treated using a compound, pharmaceutical composition, or method provided herein. In some embodiments, the disease is a malignant tumor (e.g., lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (e.g., Merkel cell carcinoma), testicular cancer, leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma). The disease may be an autoimmune, inflammatory, malignant, infectious, metabolic, developmental, cardiovascular, liver, intestinal, endocrine, neurological, or other disease.

As used herein, the term cancer refers to all types of cancers, neoplasias, or cancers found in mammals, including leukemias, lymphomas, melanomas, neuroendocrine tumors, carcinomas, and sarcomas. Exemplary cancers that can be treated using a compound, pharmaceutical composition, or method provided herein include lymphoma, sarcoma, bladder cancer, bone cancer, brain tumor, cervical cancer, colon cancer, esophageal cancer, gastric cancer. , head and neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer (eg, triple negative, ER positive, ER negative, chemotherapy resistant, Herceptin resistant, HER2 positive, doxorubicin-resistant, tamoxifen-resistant, ductal carcinoma, lobular carcinoma, primary, metastatic), ovarian cancer, pancreatic cancer, liver cancer (eg, hepatic cell carcinoma), lung cancer (eg, non-small cell lung carcinoma, squamous cell lung carcinoma, adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma, carcinoid, sarcoma), glioblastoma multiforme, glioma, melanoma, cancer

- 14 042795 prostate, castration-resistant prostate cancer, breast cancer, triple-negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma (eg head, neck or esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B-cell lymphoma, or multiple myeloma. Additional examples include thyroid, endocrine, brain, breast, cervix, colon, head and neck, esophagus, liver, kidney, lung, non-small cell lung cancer, melanoma, mesothelioma, ovarian cancer, sarcoma, malignant tumor of the stomach, uterus or medulloblastoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, malignant tumor, malignant insulinoma of the pancreas, malignant carcinoid , bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, malignant tumor of the genitourinary tract, malignant hypercalcemia, endometrial cancer, malignant tumor of the adrenal cortex, neoplasia of the endocrine or exocrine pancreas, medullary thyroid cancer melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, Paget's disease of the nipple, phyllodes tumors, lobular carcinoma, ductal carcinoma, pancreatic stellate cell malignancy, hepatic stellate cell malignancy, or prostate cancer glands.

The term leukemia broadly refers to progressive, malignant diseases of the hematopoietic organs and is generally characterized by impaired proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified based on:

(1) the duration and nature of the illness, whether acute or chronic;

(2) the type of cells affected; myeloid (myelogenous), lymphoid (lymphogenic) or monocytic; and (3) an increase or no increase in abnormal cells or non-leukemic (sub-leukemic).

Exemplary leukemias that can be treated using a compound, pharmaceutical composition, or method provided herein include, for example, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, non-leukemic leukemia, leukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia skin lesions, fetal leukemia, eosinophilic leukemia, Gross leukemia, hairy cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, leukemia with lymphosarcoma cells, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasmacytic leukemia, promyelocytic leukemia, Reeder's cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, or undifferentiated cell leukemia.

The term sarcoma generally refers to a tumor that is composed of a substance similar to embryonic connective tissue and generally consists of highly dense cells embedded in a fibrous or homogeneous substance. Sarcomas that can be treated using a compound, pharmaceutical composition, or method provided herein include chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemeti's sarcoma, fat sarcoma, liposarcoma, soft tissue alveolar sarcoma, ameloblastosarcoma, botryoid sarcoma, chloromasarcoma, choriocarcinoma, embryonic sarcoma, Wilms' sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblast sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic pigmented hemorrhagic multiple sarcoma, immunoblastic sarcoma, B cell sarcoma cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymal sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous' sarcoma, serocystic sarcoma, synovial sarcoma, or telangiectatic sarcoma.

The term melanoma has been adopted to refer to a tumor arising from the melanocytic system of the skin and other organs. Melanomas that can be treated using a compound, pharmaceutical composition, or method provided herein include, for example, acral lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, melanoma

- 15 042795 Clodmann's lanoma, S91 melanoma, Harding-Passie melanoma, juvenile melanoma, lentiginous malignant melanoma, malignant melanoma, nodular melanoma, subungual melanoma or superficially advanced melanoma.

The term carcinoma refers to a malignant new growth of epithelial cells that tends to infiltrate surrounding tissues and give rise to metastases. Exemplary carcinomas that can be treated using a compound, pharmaceutical composition, or method provided herein include, for example, medullary thyroid carcinoma, familial medullary thyroid carcinoma, acinar carcinoma, acinous carcinoma, adenoid cystic carcinoma, adenoid cystic carcinoma, adenomatous carcinoma , adrenocortical carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, basal cell carcinoma, basaloid carcinoma, basal squamous cell carcinoma, bronchoalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, medulla carcinoma, cholangiocellular carcinoma, choriocarcinoma, colloid carcinoma, acneiform carcinoma, ethmoidal carcinoma, sclerotic induration carcinoma, cutaneous carcinoma, columnar carcinoma, columnar cell carcinoma, ductal carcinoma, solid carcinoma, embryonic carcinoma, encephaloid carcinoma, epidermoid carcinoma, epithelial adenoid carcinoma, exophytic carcinoma, ulcerative carcinoma, fibrous carcinoma, gelatinous carcinoma, giant cell carcinoma, giant cell carcinoma, glandular carcinoma, granulosa cell carcinoma, hair matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hürthle cell carcinoma, hyaline carcinoma, hypernephroid carcinoma, neonatal embryonic carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, carcinoma Krompecher, Kulczycki cell carcinoma, large cell carcinoma, lenticular carcinoma, lenticular carcinoma, lipomatous carcinoma, lobular carcinoma, lymphoepithelial carcinoma, medullary cell carcinoma, medullary carcinoma, melanotic carcinoma, soft carcinoma, mucinous carcinoma, mucus-forming carcinoma, mucocellular carcinoma, mucocellular carcinoma mucosal carcinoma, mucosal carcinoma, myxomatous carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, spiny epidermocyte carcinoma, macerated carcinoma, renal cell carcinoma of the kidney, reserve cell carcinoma, sarcomoma-like carcinoma nasal mucosa, scirrhous carcinoma, scrotal carcinoma, signet cell carcinoma, simple carcinoma, small cell carcinoma, solanoid carcinoma, spherical cell carcinoma, spindle cell carcinoma, spongiform carcinoma, squamous carcinoma, squamous cell carcinoma, fibrocarcinoma, telangiectatic carcinoma, smooth muscle fiber carcinoma, and vascular tissue, transitional cell carcinoma, knobby carcinoma, tubular carcinoma, tuberous carcinoma, verrucous carcinoma, or villous carcinoma.

As used herein, the terms metastasis, metastatic, and metastatic cancer can be used interchangeably and refer to the spread of a proliferative disease or disorder, such as cancer, from one organ or other non-contiguous organ or body part. The malignant tumor originates in the original site, for example, the mammary gland, and this site is referred to as the primary tumor, for example, primary breast cancer. Some malignant cells in the primary tumor or site of origin acquire the ability to invade and infiltrate surrounding normal tissue at the local site and/or the ability to penetrate the walls of the lymphatic system or vascular system, circulating through the system and reaching other sites and body tissues. A second clinically detectable tumor derived from malignant cells of the primary tumor is referred to as a metastatic or secondary tumor. When malignant cells metastasize, it is assumed that the metastatic tumor and its cells are similar to the original tumor. Thus, if lung cancer metastasizes to the breast, the secondary tumor at the site of the breast is composed of abnormal lung cells, not abnormal breast cells. A secondary tumor in the breast is referred to as metastatic lung cancer. Thus, the phrase metastatic cancer refers to a disease in which the subject has or had a primary tumor and has one or more secondary tumors. The phrases non-metastatic cancer or subjects with a cancer that is not metastatic refers to diseases in which the subjects have a primary tumor but do not have any or more secondary tumors. For example, metastatic lung cancer refers to a disease in a subject with a primary lung tumor or a history of a primary lung tumor and one or more secondary tumors in a second site or multiple sites, such as in the breast.

The term associated or associated with in the context of a substance or activity or function of a substance associated with a disease (e.g., diabetes, cancer

- 16 042795 (for example, prostate cancer, kidney cancer, metastatic malignant tumor, melanoma, castration-resistant prostate cancer, breast cancer, triple-negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma (for example, head , neck, or esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B-cell lymphoma, or multiple myeloma)), means that the disease (eg, lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (eg, Merkel cell carcinoma), testicular cancer, leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma) is caused by ( in whole or in part), or the symptom of a disease is caused (in whole or in part) by the substance or by the activity or function of the substance.

Humanized antibodies

A humanized antibody is a genetically modified antibody in which at least one CDR (or a functional fragment thereof) from a mouse antibody (a donor antibody that may also be from a rat, hamster, or other non-human species) is grafted into a human antibody ( acceptor antibody). A human antibody is a non-natural (e.g., not naturally occurring or not naturally produced in humans) antibody that does not elicit an immune response in a human, does not elicit a significant immune response in a human, or elicits an immune response that is less than that elicited in humans. mice. In some embodiments, more than one mouse CDR is grafted (eg, all six mouse CDRs are grafted). The acceptor antibody sequence may be, for example, a mature human antibody sequence (or fragment thereof), a consensus sequence from human antibody sequences (or fragment thereof), or a germline sequence (or fragment thereof). Thus, a humanized antibody may be an antibody having one or more CDRs from a donor antibody and a variable region (FR) framework. The FR may form part of a constant region and/or a variable region in a human antibody. In addition, to maintain high binding affinity, the amino acids in the human acceptor sequence can be replaced with the corresponding amino acids from the donor sequence, for example, where:

(1) the amino acid is in the CDR;

(2) the amino acid is in the human framework region (eg, the amino acid is in the immediate vicinity of one of the CDRs).

See US Pat. Nos. 5,530,101 and 5,585,089, incorporated herein by reference, for detailed instructions for constructing humanized antibodies. Although humanized antibodies often include all six CDRs (e.g., as defined by Kabat, but often also including the H1 hypervariable loop, as defined by Chothia) from a mouse antibody, they can also be made with fewer mouse CDRs and/or less than complete mouse CDR sequence (e.g., a functional CDR fragment) (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al. , Mol Immunol 36:1079-1091, 1999; Tamura et al., Journal of Immunology, 164:1432-1441, 2000).

Typically, a humanized antibody as described herein may contain:

(i) a light chain comprising at least one CDR (often three CDRs) from a mouse antibody (also referred to herein as mouse CDRs) and a human variable region framework; and (ii) a heavy chain containing at least one CDR (often three CDRs) from a mouse antibody and a human variable region (FR) framework.

Each of the light and heavy chain variable region (FR) frameworks can be a mature human antibody variable region framework sequence (or a fragment thereof), a germline variable region framework sequence (in combination with a J region) (or a fragment thereof), or a consensus sequence from human antibody variable region framework sequences (or a fragment thereof). In some embodiments, the humanized antibody comprises a light chain as described in (i), a heavy chain as described in (ii), together with a human light chain constant region and a heavy chain constant region.

A chimeric antibody is an antibody in which the variable region of a mouse (or other rodent) antibody is combined with a constant region of a human antibody; their design by genetic engineering is well known. Such antibodies retain the binding specificity of the mouse antibody while being about two-thirds human. The proportion of non-human sequences present in mouse, chimeric and humanized antibodies suggests that the immunogenicity of chimeric antibodies is intermediate between mouse and humanized antibodies. Other types of genetically modified antibodies that may have reduced immunogenicity compared to murine antibodies include human antibodies generated using phage display techniques.

- 17 042795 (Dower et al., WO 91/17271; McCafferty et al., WO 92/001047; Winter, WO 92/20791 and Winter, FEBS Lett. 23:92, 1998, the content of each of which is given in this description as a reference) or using transgenic animals (Lonberg et al., WO 93/12227; Kucherlapati WO 91/10741, the content of each of which is given in the present description by reference).

Other methods for developing humanized antibodies can also be used to achieve the same results as with the methods of US Patent No. 5530101 and 5585089 described above, for example, superhumanization (see Tan et al., J. Immunol. 169: 1119, 2002 and US Patent No. 6881557) or with the method of Studnicak et al., Protein Eng. 7:805, 1994. In addition, other methods for producing genetically modified mAbs with reduced immunogenicity include structural rearrangement, hyperchimerization, and cladding/surfacing, as described, for example, in Vaswami et al., Annals of Allergy, Asthma and Immunology 81:105 , 1998; Roguska et al., Protein Eng. 9:895, 1996 and US Patent Nos. 6,072,035 and 5,639,641.

The CD73 protein or CD73 antigen as defined herein includes any of the recombinant or natural forms of the 73 cassette of differentiation (CD73), also known as 5'-nucleotidase (5'-NT) or ecto-5'-nucleotidase, or variants thereof or homologues that retain the nucleotidase activity of CD73 (eg, within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to CD73). In some aspects, variants or homologues have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of a sequence (e.g., portions of 50, 100, 150, or 200 contiguous amino acids) at compared with the natural protein CD73. In some embodiments, the CD73 protein is substantially identical to the protein identified by reference number in UniProt 21589, or to a variant or homologue that has significant identity therewith. In some embodiments, the CD73 protein is substantially identical to the protein identified under UniProt reference number Q61503 or a variant or homologue that has significant identity therewith.

Humanized antibodies 1E9

The present invention relates, inter alia, to humanized 1E9 antibodies containing a humanized light chain variable region and a humanized heavy chain variable region. Humanized 1E9 antibodies as described herein are capable of binding the CD73 protein and contain at least one CDR or functional fragment thereof from the mouse monoclonal antibody 1E9 (Thomson LF et al., Tissue Antigens 2008, Volume 35, Issue 1: Production and characterization of monoclonal antibodies to the glycosyl phosphatidylinositol-anchored lymphocyte differentiation antigen ecto-5'-nucleotidase (CD73)).

A functional CDR fragment is a portion of the complete amino acid sequence of a CDR that is capable of binding to an antigen (eg, CD73). Thus, a functional fragment of a CDR will typically include the amino acid residues required for the CDR to bind to an antigen (eg, CD73). A mouse CDR is the complete amino acid sequence of a CDR, or a functional fragment thereof, derived from a mouse antibody capable of binding CD73. Thus, a functional fragment of a murine CDR typically includes the amino acid residues required for CDR binding to CD73. When the humanized 1E9 antibody includes at least one murine CDR, at least one murine CDR or functional fragment thereof is derived from the donor antibody. In some embodiments, the donor antibody is a mouse 1E9 antibody. One of skill in the art will immediately recognize that a humanized 1E9 antibody containing at least one mouse CDR is a humanized antibody with at least one mouse CDR derived from the donor 1E9 antibody and additional CDRs derived from the acceptor antibody (e.g., where light the chain has only three CDRs, and the heavy chain has only three CDRs).

In some embodiments, the humanized light chain variable region and the humanized heavy chain variable region collectively comprise one mouse CDR or a functional fragment of a mouse CDR. Thus, in some embodiments, the humanized light chain variable region and the humanized heavy chain variable region comprise six CDRs in total, wherein at least one of the six CDRs is a murine CDR. When the humanized light chain variable region and the humanized heavy chain variable region comprise one murine CDR together, the humanized light chain variable region or the humanized heavy chain variable region contains one murine CDR. For example, a humanized antibody may include an L3 CDR derived from a donor antibody (e.g., a mouse, also referred to herein as mouse CDR L3) and an L1 CDR, L2 CDR, H1 CDR, H2 CDR, and H3 CDR derived from an acceptor antibody (i.e., a mouse CDR). .e. human).

In some embodiments, the humanized light chain variable region and the humanized heavy chain variable region comprise two murine CDRs in combination. When the variable region of the humanized light chain and the variable region of the humanized strand

- 18 042795 of the gel chain comprise two murine CDRs in total, each of the humanized light chain variable region and the humanized heavy chain variable region contains one murine CDR (i), the humanized light chain variable region contains two murine CDRs (ii), or the humanized heavy chain variable region the heavy chain contains two mouse CDRs (iii). For example, a humanized antibody may include an L3 CDR and an H3 CDR derived from a donor antibody (also referred to herein as mouse L3 CDR and mouse H3 CDR, respectively) and an L1 CDR, L2 CDR, H1 CDR, and H2 CDR derived from an acceptor antibody. antibodies (i.e. human).

In some embodiments, the humanized light chain variable region and the humanized heavy chain variable region comprise three murine CDRs in total. When the humanized light chain variable region and the humanized heavy chain variable region comprise three murine CDRs in total, the humanized light chain variable region may contain one murine CDR, and the humanized heavy chain variable region may comprise two murine CDRs (i), the humanized light chain variable region contains two mouse CDRs and the humanized heavy chain variable region contains one mouse CDR (ii), the humanized light chain variable region contains three mouse CDRs (iii), or the humanized heavy chain variable region contains three mouse CDRs (iv). For example, a humanized antibody may include CDR L3, CDR H3, and CDR L2 derived from a donor antibody (e.g., murine, also referred to herein as CDR L3, CDR H3 mouse, and CDR L2 mouse, respectively) and CDR L1, CDR H1, and H2 CDRs derived from an acceptor antibody (ie, human).

In some embodiments, the humanized light chain variable region and the humanized heavy chain variable region comprise four murine CDRs in total. When the humanized light chain variable region and the humanized heavy chain variable region comprise four murine CDRs in total, the humanized light chain variable region contains one murine CDR, and the humanized heavy chain variable region contains three murine CDRs (i), the humanized light chain variable region contains three mouse CDRs and the humanized heavy chain variable region contains one mouse CDR (ii), or the humanized light chain variable region contains two mouse CDRs and the humanized heavy chain variable region contains two mouse CDRs (iii). For example, a humanized antibody may include CDR L3, CDR H3, CDR L2, and CDR L1 derived from a donor antibody (e.g., murine, also referred to herein as mouse CDR L3, mouse CDR H3, mouse CDR L2, and mouse CDR L1, respectively) and CDR H1 and CDR H2 derived from an acceptor antibody (ie, human).

In some embodiments, each of the humanized light chain variable region and the humanized heavy chain variable region includes at least one murine CDR. When the humanized light chain variable region and the humanized heavy chain variable region each comprise at least one mouse CDR, the humanized light chain variable region contains at least one mouse CDR and the humanized heavy chain variable region contains at least one mouse CDR. Thus, in some embodiments, the humanized light chain variable region contains a mouse L1 CDR and the humanized heavy chain contains a mouse H1 CDR. In some embodiments, the mouse L1 CDR comprises the amino acid sequence from SEQ ID NO: 1 and the mouse CDR H1 includes the amino acid sequence from SEQ ID NO: 4. In some embodiments, the mouse L1 CDR is the amino acid sequence from SEQ ID NO: 1 and the H1 CDR mouse is the amino acid sequence of SEQ ID NO: 4. In some embodiments, the humanized light chain variable region contains a mouse L2 CDR and the humanized heavy chain variable region contains a mouse H2 CDR. In some embodiments, the mouse L2 CDR comprises the amino acid sequence from SEQ ID NO: 2 and the mouse CDR H2 includes the amino acid sequence from SEQ ID NO: 5. In some embodiments, the mouse L2 CDR is the amino acid sequence from SEQ ID NO: 2 and the H2 CDR mouse is the amino acid sequence of SEQ ID NO: 5. In some embodiments, the humanized light chain variable region contains a mouse L3 CDR and the humanized heavy chain variable region contains a mouse H3 CDR. In some embodiments, the mouse L3 CDR comprises the amino acid sequence from SEQ ID NO: 3 and the mouse H3 CDR includes the amino acid sequence from SEQ ID NO: 6. In some embodiments, the L3 CDR is the amino acid sequence from SEQ ID NO: 3 and the mouse H3 CDR is the amino acid sequence from SEQ ID NO: 6.

In some embodiments, the presence of a mouse L3 CDR and a mouse H3 CDR may be sufficient for the humanized antibody to bind to CD73. Thus, in some embodiments, the humanized antibody does not include a mouse L1 CDR, a mouse L2 CDR, a mouse H1 CDR, or a mouse H2 CDR. When a humanized antibody does not include a mouse L1 CDR, a mouse L2 CDR, a mouse H1 CDR, or a mouse H2 CDR, the humanized antibody includes a CDR L1, CDR L2, CDR H1, or CDR

- 19 042795

H2 derived from an acceptor antibody (i.e. human). Thus, a humanized antibody that does not include a mouse L1 CDR, a mouse L2 CDR, a mouse H1 CDR, or a mouse H2 CDR does not include an L1 CDR, L2 CDR, H1 CDR, or H2 CDR from a donor antibody (e.g., mouse, rat, rabbit) but includes an L1 CDR, an L2 CDR, an H1 CDR, or an H2 CDR from an acceptor antibody (i.e., human). Thus, in some embodiments, the humanized light chain variable region does not include a mouse L1 CDR or mouse L2 CDR, and the humanized heavy chain variable region does not include a mouse H1 CDR or mouse H2 CDR. In some embodiments, the humanized light chain variable region does not include mouse L1 CDRs and mouse L2 CDRs, and the humanized heavy chain variable region does not include mouse H1 CDRs and mouse H2 CDRs.

In some embodiments, the humanized light chain variable region comprises a mouse L2 CDR and a mouse L3 CDR, and the humanized heavy chain variable region comprises a mouse H2 CDR and a mouse H3 CDR. In some embodiments, the humanized light chain variable region comprises a mouse CDR of L1, a mouse CDR of L2, and a mouse CDR of L3, and the humanized heavy chain variable region comprises a mouse CDR of H1, a mouse CDR of H2, and a mouse CDR of H3. In some embodiments, the humanized light chain variable region comprises a mouse L1 CDR as listed in SEQ ID NO: 1, a mouse L2 CDR as listed in SEQ ID NO: 2, and a mouse L3 CDR as listed in SEQ ID NO: 3, and the humanized heavy chain variable region comprises a mouse H1 CDR as set forth in SEQ ID NO: 4, a mouse H2 CDR as set forth in SEQ ID NO: 5, and a mouse H3 CDR as set forth in SEQ ID NO: 6.

The position of the CDR and FR can be determined by the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, U.S. Government Printing Office (1991)). Similarly, the positions occupied by individual residues in the light or heavy chain of an antibody can be determined by the Kabat numbering system. Thus, the location of the residues required for binding in the humanized light chain and in the humanized heavy chain of a humanized antibody can be determined by the position of the residue according to the Kabat numbering system, as is well known in the art. As described above, a humanized antibody can be an antibody having a CDR from a donor antibody (eg, a mouse) and a variable region (FR) framework from a human antibody. The framework regions (FRs) are believed to hold the CDRs in place in the humanized antibody. Starting at the amino terminus, these regions are designated FR L1, FR L2, FR L3 and FR L4 for the light chain and FR H1, FR H2, FR H3 and FR H4 for the heavy chain, respectively. Paradoxically, the present invention relates to humanized antibodies comprising one or more residues within framework regions that are important for the binding of the humanized antibody to an epitope. A framework region residue involved in binding (or important for binding) an epitope (eg, CD73 binding) is referred to herein as a binding framework residue. Framework binding residues may reside within the humanized light chain variable region framework (i.e., FR L1, FR L2, FR L3, FR L4) or they may reside within the humanized heavy chain variable region framework (i.e., FR H1 , FR H2, FR H3, FR H4). The binding residue of the framework region located in the FR L3 region of the humanized light chain is referred to herein as the binding residue of the FR L3 framework region. Thus, the binding residue of the framework region located in the FR H3 region of the humanized heavy chain is referred to herein as the binding residue of the FR H3 framework region.

In some embodiments, the implementation of the humanized antibody contains at least one binding residue of the frame region. In some embodiments, the humanized light chain variable region contains at least one framework binding residue. In some embodiments, the humanized light chain variable region comprises one or more FR L1, FR L2, FR L3, or FR L4 framework binding residues. In some embodiments, the humanized light chain variable region contains one or more L1 FR framework binding residues. In some embodiments, the humanized light chain variable region contains one or more L2 FR framework binding residues. In some embodiments, the humanized light chain variable region contains one or more L3 FR framework binding residues. In some embodiments, the humanized light chain variable region contains one or more L4 FR framework binding residues. In some embodiments, the humanized heavy chain variable region comprises one or more FR H1, FR H2, FR H3, or FR H4 framework region binding residues. In some embodiments, the humanized heavy chain variable region contains one or more H1 FR framework binding residues. In some embodiments, the humanized heavy chain variable region contains one or more H2 FR framework binding residues. In some embodiments, the humanized heavy chain variable region contains one or more H3 FR framework binding residues. In some embodiments, the humanized heavy chain variable region comprises

- 20 042795 one or more binding residues of the frame region FR H4.

In some embodiments, the humanized light chain variable region contains at least one framework binding residue (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 37, 38, 39, 40, 41, 42 43, 44, 45, 46, 47, 48, 49, 50 or more residues), and the humanized heavy chain variable region contains at least one framework binding residue (e.g., 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 , 31, 32, 33, 34, 35, 36 37, 38, 39, 40, 41, 42 43, 44, 45, 46, 47, 48, 49, 50 or more residues). The position of the binding residue of the framework region in the humanized antibody can be determined by the Kabat numbering system, similar to the positions of the CDR residues.

In one aspect, the present invention provides a humanized 1E9 antibody comprising a humanized light chain variable region, including a mouse CDR L1, a mouse CDR L2, or a mouse CDR L3, and a humanized heavy chain variable region, including a mouse CDR H1, a mouse CDR H2, or a mouse H3 CDR. . The humanized light chain variable region may include a mouse CDR L1 as set forth in SEQ ID NO: 1, a mouse CDR L2 as set forth in SEQ ID NO: 2, or a mouse CDR L3 as set forth in SEQ ID NO: 3. The light chain may include a mouse L1 CDR as set forth in SEQ ID NO: 1, a mouse L2 CDR as set forth in SEQ ID NO: 2, and a mouse L3 CDR as set forth in SEQ ID NO: 3. A humanized heavy chain variable region may include a mouse H1 CDR as listed in SEQ ID NO: 4, a mouse H2 CDR as listed in SEQ ID NO: 5, or a mouse H3 CDR as listed in SEQ ID NO: 6. The humanized heavy chain variable region may include CDR H1 mouse as indicated in SEQ ID NO: 4, mouse H2 CDR as indicated in SEQ ID NO: 5, and mouse H3 CDR as indicated in SEQ ID NO: 6. In some embodiments, the humanized light chain variable region comprises CDR L1 mouse as set forth in SEQ ID NO: 1. In some embodiments, the humanized light chain variable region comprises a mouse L2 CDR as set forth in SEQ ID NO: 2. In some embodiments, the humanized light chain variable region comprises a mouse L3 CDR as set forth in SEQ ID NO: 3. In some embodiments, the humanized heavy chain variable region comprises a mouse H1 CDR as set forth in SEQ ID NO: 4. In some embodiments, the humanized heavy chain variable region comprises a mouse H2 CDR as set forth in SEQ ID NO: : 5. In some embodiments, the humanized light chain variable region comprises a mouse H3 CDR as indicated in SEQ ID NO: 6. In further embodiments, the humanized light chain variable region comprises at least one framework binding residue. In other additional embodiments, the implementation of the variable region of the humanized heavy chain contains at least one binding framework region residue.

In one aspect, the present invention provides a humanized 1E9 antibody. The 1E9 antibody includes a humanized light chain variable region and a humanized heavy chain variable region. The variable region of the humanized light chain contains:

(i) mouse CDR L1 as listed in SEQ ID NO: 1, mouse CDR L2 as listed in SEQ ID NO: 2, mouse CDR L3 as listed in SEQ ID NO: 3, and (ii) valine at the position, Kabat position 2, methionine at Kabat position 4, aspartic acid or leucine at Kabat position 9, proline or serine at Kabat position 12, lysine or proline at position 18 Kabat, alanine at Kabat position 43, proline or serine at Kabat position 60, threonine at Kabat position 74, asparagine or serine at Kabat position 76, asparagine or serine at Kabat position 77, isoleucine or leucine at Kabat position 78, serine or alanine at Kabat position 80, glutamine at Kabat position 100, valine at Kabat position 104 Kabat, glutamic acid or alanine at Kabat position 1, glutamine at Kabat position 3, phenylalanine or threonine at Kabat position 10, glutamine at Kabat position 11, alanine or leucine at Kabat position 13, threonine at Kabat position 14, valine or proline at Kabat position 15, lysine at Kabat position 16, glutamic acid or aspartic acid at position, Kabat position 17, threonine at Kabat position 22, lysine at Kabat position 42, arginine at Kabat position 45, isoleucine at Kabat position 58, tyrosine at Kabat position 67, phenylalanine at Kabat position 73, tyrosine at Kabat position 85, or phenylalanine at Kabat position 87.

- 21 042795

The variable region of a humanized heavy chain contains:

(i) mouse H1 CDR as set out in SEQ ID NO: 4, mouse H2 CDR as set out in SEQ ID NO: 5, and mouse H3 CDR as set out in SEQ ID NO: 6, and (ii) isoleucine at position , corresponding to Kabat position 37, alanine or proline in position corresponding to Kabat position 40, lysine in position corresponding to Kabat position 4, serine in position corresponding to Kabat position 70, isoleucine or threonine in position corresponding to Kabat position 75 , tryptophan at Kabat position 82, arginine or lysine at Kabat position 83, alanine at Kabat position 84, serine at Kabat position 85, valine or methionine at position Kabat position 89, valine at Kabat position 5, serine at Kabat position 7, valine at Kabat position 11, glutamic acid or lysine at Kabat position 12, isoleucine or valine at Kabat position 20, arginine at Kabat position 38, arginine at Kabat position 66, valine at Kabat position 67, isoleucine at Kabat position 69, alanine at Kabat position 71, lysine at Kabat position 73, threonine at Kabat position 87, glutamic acid at Kabat position 1, valine at Kabat position 24 , arginine at Kabat position 44, methionine at Kabat position 48, leucine at Kabat position 80, or glutamic acid at Kabat position 81.

In some embodiments, the humanized light chain variable region provided herein comprises a framework binding residue that is a valine at Kabat position 2, a methionine at Kabat position 4, an aspartic acid, or a leucine at Kabat position 9, proline or serine at Kabat position 12, lysine or proline at Kabat position 18, alanine at Kabat position 43, proline or serine at Kabat position 60 Kabat, threonine at Kabat position 74, asparagine or serine at Kabat position 76, asparagine or serine at Kabat position 77, isoleucine or leucine at Kabat position 78, serine or alanine at Kabat position 80, glutamine at Kabat position 100, valine at Kabat position 104, glutamic acid or alanine at Kabat position 1, glutamine at position 3 Kabat, phenylalanine or threonine at Kabat position 10, glutamine at Kabat position 11, alanine or leucine at Kabat position 13, threonine at Kabat position 14, valine or proline in Kabat position 15, lysine in Kabat position 16, glutamic acid or aspartic acid in Kabat position 17, threonine in Kabat position 22, lysine in position 42 Kabat, arginine at position 45 Kabat, isoleucine at position 58 Kabat, tyrosine at position 67 Kabat, phenylalanine at position 73 Kabat, tyrosine at position 85 according to Kabat, or phenylalanine in the position corresponding to position 87 according to Kabat.

In some embodiments, the humanized light chain variable region contains a framework region binding residue that is a valine at a position corresponding to Kabat position 2. In some embodiments, the humanized light chain variable region contains a framework region binding residue that is a methionine at a position corresponding to position 4 according to Kabat. In some embodiments, the humanized light chain variable region comprises an aspartic acid or leucine framework binding residue at a position corresponding to Kabat position 9. In some embodiments, the humanized light chain variable region contains a framework binding residue that is proline or serine at position corresponding to Kabat position 12. In some embodiments, the humanized light chain variable region contains a framework binding residue that is a lysine or a proline at a position corresponding to Kabat position 18. In some embodiments, the humanized light chain variable region contains a framework region binding residue that is an alanine at a position corresponding to Kabat position 43. In some embodiments, the variable region is humanized

- 22 042795 The light chain bath contains a binding residue of the framework region, which is a proline or serine in the position corresponding to position 60 according to Kabat.

In some embodiments, the humanized light chain variable region contains a framework binding residue that is threonine at a position corresponding to Kabat position 74. In some embodiments, the humanized light chain variable region contains an asparagine or serine binding framework residue at position corresponding to Kabat position 76. In some embodiments, the humanized light chain variable region contains an asparagine or serine binding framework region at position corresponding to Kabat position 77. In some embodiments, the humanized light chain variable region contains an isoleucine or leucine binding framework region at position corresponding to Kabat position 78. In some embodiments, the humanized light chain variable region contains a framework region binding residue that is serine or alanine at position corresponding to Kabat position 80. In some embodiments, the humanized light chain variable region contains a framework binding residue that is glutamine at position corresponding to Kabat position 100. In some embodiments, the humanized light chain variable region contains a framework region binding residue that is a valine at a position corresponding to Kabat position 104. In some embodiments, the humanized light chain variable region contains a framework region binding residue that is glutamic acid or alanine at a position corresponding to Kabat position 1. In some embodiments, the humanized light chain variable region contains a framework binding residue that is glutamine at a position corresponding to Kabat position 3.

In some embodiments, the humanized light chain variable region contains a framework binding residue that is phenylalanine or threonine at a position corresponding to Kabat position 10. In some embodiments, the humanized light chain variable region contains a framework binding residue that is glutamine at a position corresponding to Kabat position 11. In some embodiments, the humanized light chain variable region contains an alanine or leucine binding framework residue at position corresponding to Kabat position 13. In some embodiments, the humanized light chain variable region contains a framework region binding residue that is threonine at position corresponding to Kabat position 14. In some embodiments, the humanized light chain variable region contains a framework region binding residue that is a valine or proline at a position corresponding to Kabat position 15. In some embodiments, the humanized light chain variable region contains a framework binding residue that is a lysine at a position corresponding to Kabat position 16. In some embodiments, the humanized light chain variable region contains a framework binding residue that is glutamic acid or aspartic acid at position corresponding to Kabat position 17. In some embodiments, the humanized light chain variable region contains a framework binding residue that is threonine at a position corresponding to Kabat position 22.

In some embodiments, the humanized light chain variable region contains a framework binding residue that is a lysine at a position corresponding to Kabat position 42. In some embodiments, the humanized light chain variable region contains an arginine framework binding residue at position corresponding to Kabat position 45. In some embodiments, the humanized light chain variable region contains a framework binding residue that is isoleucine at a position corresponding to Kabat position 58. In some embodiments, the humanized light chain variable region contains a framework region binding residue that is a tyrosine at a position corresponding to Kabat position 67. In some embodiments, the humanized light chain variable region contains a framework binding residue that is phenylalanine at the position corresponding to position 73 according to Kabat. In some embodiments, the humanized light chain variable region contains an isoleucine framework binding residue at position corresponding to Kabat position 78. In some embodiments, the humanized light chain variable region contains a framework region binding residue that is a tyrosine at a position corresponding to Kabat position 85. In some embodiments, the humanized light chain variable region contains a framework binding residue that is a phenylalanine at position

- 23 042795 nii, corresponding to the provision 87 according to Kabat.

The humanized heavy chain variable region provided herein may include a framework region binding residue that is isoleucine at Kabat position 37, alanine or proline at Kabat position 40, lysine at position 43 Kabat, serine at Kabat position 70, isoleucine or threonine at Kabat position 75, tryptophan at Kabat position 82, arginine or lysine at Kabat position 83, alanine at Kabat position 84, serine at Kabat position 85, valine or methionine at Kabat position 8 9, valine at Kabat position 5, serine at Kabat position 7, valine at position 11 Kabat, glutamic acid or lysine at position 12 Kabat, isoleucine or valine at position 20 Kabat, arginine at position 38 Kabat, arginine at position Kabat 66, valine at Kabat 67, isoleucine at Kabat 69, alanine at Kabat 71, lysine at Kabat 73, threonine at Kabat 87 by Kabat, glutamic acid at position corresponding to position 1 by Kabat, valine at position corresponding to position 24 by Kabat, arginine at position corresponding to position 44 by Kabat, methionine at position corresponding to position 48 by Kabat, leucine at position corresponding to position 80 according to Kabat, or glutamic acid in the position corresponding to position 81 according to Kabat.

In some embodiments, the humanized heavy chain variable region contains a framework binding residue that is isoleucine at a position corresponding to Kabat position 37. In some embodiments, the humanized heavy chain variable region contains a framework region binding residue that is an alanine or a proline at a position corresponding to position 40 according to Kabat. In some embodiments, the humanized heavy chain variable region contains a framework binding residue that is a lysine at a position corresponding to Kabat position 43. In some embodiments, the humanized heavy chain variable region contains a framework binding residue that is serine at position corresponding to position 70 according to Kabat. In some embodiments, the humanized heavy chain variable region contains an isoleucine or threonine binding framework region residue at a position corresponding to Kabat position 75. In some embodiments, the humanized heavy chain variable region contains a tryptophan binding framework residue at position corresponding to Kabat position 82. In some embodiments, the humanized heavy chain variable region contains an arginine or lysine binding framework region residue at a position corresponding to Kabat position 83. In some embodiments, the humanized heavy chain variable region contains a framework region binding residue that is an alanine at a position corresponding to Kabat position 84.

In some embodiments, the humanized heavy chain variable region contains a framework binding residue that is serine at a position corresponding to Kabat position 85. In some embodiments, the humanized heavy chain variable region contains a framework region binding residue that is a valine or methionine at a position corresponding to Kabat position 89. In some embodiments, the humanized heavy chain variable region contains a framework region binding residue that is a valine at a position corresponding to Kabat position 5. In some embodiments, the humanized heavy chain variable region contains a framework binding residue that is serine at a position corresponding to Kabat position 7. In some embodiments, the humanized heavy chain variable region contains a framework region binding residue that is a valine at a position corresponding to Kabat position 11. In some embodiments, the humanized heavy chain variable region contains a framework binding residue that is glutamic acid or lysine at a position corresponding to Kabat position 12. In some embodiments, the humanized heavy chain variable region comprises an isoleucine or valine binding framework region at position corresponding to Kabat position 20. In some embodiments, the humanized heavy chain variable region contains an arginine binding framework region residue at a position corresponding to Kabat position 38. In some embodiments, the humanized heavy chain variable region contains a framework region binding residue that is arginine at a position corresponding to

- 24 042795 position 66 of Kabat. In some embodiments, the humanized heavy chain variable region contains a framework region binding residue that is a valine at a position corresponding to Kabat position 67.

In some embodiments, the humanized heavy chain variable region contains a framework binding residue that is isoleucine at a position corresponding to Kabat position 69. In some embodiments, the humanized heavy chain variable region contains a framework region binding residue that is an alanine at a position corresponding to Kabat position 71. In some embodiments, the humanized heavy chain variable region contains a framework binding residue that is a lysine at a position corresponding to Kabat position 73. In some embodiments, the humanized heavy chain variable region contains a framework binding residue that is threonine at a position corresponding to Kabat position 87. In some embodiments, the humanized heavy chain variable region contains a framework binding residue that is glutamic acid at a position corresponding to Kabat position 1. In some embodiments, the humanized heavy chain variable region contains a framework region binding residue that is a valine at a position corresponding to Kabat position 24. In some embodiments, the humanized heavy chain variable region contains a framework binding residue that is arginine at a position corresponding to Kabat position 44. In some embodiments, the humanized heavy chain variable region contains a framework region binding residue that is a methionine at a position corresponding to Kabat position 48. In some embodiments, the humanized heavy chain variable region contains a framework binding residue that is leucine at position corresponding to Kabat position 80. In some embodiments, the humanized heavy chain variable region contains a framework binding residue that is glutamic acid at a position corresponding to Kabat position 81.

In some embodiments, the humanized light chain variable region comprises valine at Kabat position 2, methionine at Kabat position 4, leucine at Kabat position 9, proline at Kabat position 12, or proline at position corresponding to position 18 according to Kabat; and the humanized heavy chain variable region contains isoleucine at Kabat position 37, proline at Kabat position 40, lysine at Kabat position 43, serine at Kabat position 70, isoleucine at position , Kabat position 75, tryptophan at Kabat position 82, lysine at Kabat position 83, alanine at Kabat position 84, serine at Kabat position 85, or methionine at a provision corresponding to regulation 89 of Kabat.

In some embodiments, the humanized light chain variable region comprises valine at Kabat position 2, methionine at Kabat position 4, leucine at Kabat position 9, proline at Kabat position 12, and proline at position corresponding to position 18 according to Kabat; and the humanized heavy chain variable region contains isoleucine at Kabat position 37, proline at Kabat position 40, lysine at Kabat position 43, serine at Kabat position 70, isoleucine at position , Kabat position 75, tryptophan at Kabat position 82, lysine at Kabat position 83, alanine at Kabat position 84, serine at Kabat position 85, or methionine at a provision corresponding to regulation 89 of Kabat.

In some embodiments, the humanized light chain variable region comprises valine at Kabat position 2, methionine at Kabat position 4, leucine at Kabat position 9, proline at Kabat position 12, or proline at position corresponding to position 18 according to Kabat; and the humanized heavy chain variable region contains isoleucine at Kabat position 37, proline at Kabat position 40, lysine at Kabat position 43, serine at Kabat position 70, isoleucine at position , corresponding to Kabat position 75, tryptophan at position corresponding to Kabat position 82, lysine at position corresponding to Kabat position 83, alanine at position corresponding to Kabat position 84, serine at position corresponding to Kabat position 85, and methionine at position corresponding to

- 25 042795 position 89 of Kabat.

In some embodiments, the humanized light chain variable region comprises valine at Kabat position 2, methionine at Kabat position 4, leucine at Kabat position 9, proline at Kabat position 12, and proline at position corresponding to position 18 according to Kabat; and the humanized heavy chain variable region contains isoleucine at Kabat position 37, proline at Kabat position 40, lysine at Kabat position 43, serine at Kabat position 70, isoleucine at position , corresponding to Kabat position 75, tryptophan at position corresponding to Kabat position 82, lysine at position corresponding to Kabat position 83, alanine at position corresponding to Kabat position 84, serine at position corresponding to Kabat position 85, and methionine at a provision corresponding to regulation 89 of Kabat.

In some embodiments, the humanized light chain variable region comprises proline or serine at position corresponding to Kabat position 12, alanine at position corresponding to Kabat position 43, proline or serine at position corresponding to Kabat position 60, threonine at position corresponding to position 74 Kabat, asparagine or serine at position 76 Kabat, asparagine or serine at position 77 Kabat, isoleucine or leucine at position 78 Kabat, serine or alanine at position 80 Kabat, glutamine at Kabat position 100 or valine at Kabat position 104; and the humanized heavy chain variable region comprises valine at Kabat position 5, serine at Kabat position 7, valine at Kabat position 11, glutamic acid or lysine at Kabat position 12, isoleucine or valine at Kabat position 20, arginine at Kabat position 38, alanine or proline at Kabat position 40, arginine at Kabat position 66, valine at position Kabat 67, isoleucine at Kabat position 69, alanine at Kabat position 71, lysine at Kabat position 73, isoleucine or threonine at Kabat position 75, arginine or lysine at position corresponding to position 83 according to Kabat, or threonine in position corresponding to position 87 according to Kabat.

In some embodiments, the humanized light chain variable region comprises proline or serine at position corresponding to Kabat position 12, alanine at position corresponding to Kabat position 43, proline or serine at position corresponding to Kabat position 60, threonine at position corresponding to position 74 Kabat, asparagine or serine at position 76 Kabat, asparagine or serine at position 77 Kabat, isoleucine or leucine at position 78 Kabat, serine or alanine at position 80 Kabat, glutamine at Kabat position 100 and valine at Kabat position 104; and the humanized heavy chain variable region comprises valine at Kabat position 5, serine at Kabat position 7, valine at Kabat position 11, glutamic acid or lysine at Kabat position 12, isoleucine or valine at Kabat position 20, arginine at Kabat position 38, alanine or proline at Kabat position 40, arginine at Kabat position 66, valine at position Kabat 67, isoleucine at Kabat position 69, alanine at Kabat position 71, lysine at Kabat position 73, isoleucine or threonine at Kabat position 75, arginine or lysine at position corresponding to position 83 according to Kabat or threonine in position corresponding to position 87 according to Kabat.

In some embodiments, the humanized light chain variable region comprises proline or serine at position corresponding to Kabat position 12, alanine at position corresponding to Kabat position 43, proline or serine at position corresponding to Kabat position 60, threonine at position corresponding to position 74 Kabat, asparagine or serine at position 76 Kabat, asparagine or serine at position 77 Kabat, isoleucine or leucine at position 78 Kabat, serine or alanine at position 80 Kabat, glutamine at Kabat position 100 or valine at Kabat position 104; and the variable region of the humanized heavy chain contains valine at the position

- 26 042795 Kabat position 5, serine at Kabat position 7, valine at Kabat position 11, glutamic acid or lysine at Kabat position 12, isoleucine or valine at position 20 Kabat, arginine at position 38 Kabat, alanine or proline at position 40 Kabat, arginine at position 66 Kabat, valine at position 67 Kabat, isoleucine at position Kabat position 69, alanine at Kabat position 71, lysine at Kabat position 73, isoleucine or threonine at Kabat position 75, arginine or lysine at Kabat position 83, and threonine at the position corresponding to position 87 according to Kabat.

In some embodiments, the humanized light chain variable region comprises proline or serine at position corresponding to Kabat position 12, alanine at position corresponding to Kabat position 43, proline or serine at position corresponding to Kabat position 60, threonine at position corresponding to position 74 Kabat, asparagine or serine at position 76 Kabat, asparagine or serine at position 77 Kabat, isoleucine or leucine at position 78 Kabat, serine or alanine at position 80 Kabat, glutamine at Kabat position 100 and valine at Kabat position 104; and the humanized heavy chain variable region comprises valine at Kabat position 5, serine at Kabat position 7, valine at Kabat position 11, glutamic acid or lysine at Kabat position 12, isoleucine or valine at Kabat position 20, arginine at Kabat position 38, alanine or proline at Kabat position 40, arginine at Kabat position 66, valine at position Kabat 67, isoleucine at Kabat position 69, alanine at Kabat position 71, lysine at Kabat position 73, isoleucine or threonine at Kabat position 75, arginine or lysine at position corresponding to position 83 according to Kabat, and threonine in position corresponding to position 87 according to Kabat.

In some embodiments, the humanized light chain variable region comprises glutamic acid or alanine at Kabat position 1, valine at Kabat position 2, glutamine at Kabat position 3, methionine at position 4 Kabat, aspartic acid or leucine at Kabat position 9, phenylalanine or threonine at Kabat position 10, glutamine at Kabat position 11, serine or proline at Kabat position 12, alanine or leucine at Kabat position 13, threonine at Kabat position 14, valine or proline at Kabat position 15, lysine at Kabat position 16, glutamic acid or aspartic acid at Kabat position 17, lysine or proline in Kabat position 18, threonine in Kabat position 22, lysine in Kabat position 42, arginine in Kabat position 45, isoleucine at Kabat position 58, proline or serine at Kabat position 60, tyrosine at Kabat position 67, phenylalanine at Kabat position 73, isoleucine at Kabat position 78 Kabat, serine or alanine at Kabat position 80, tyrosine at Kabat position 85, or phenylalanine at Kabat position 87; and the humanized heavy chain variable region contains glutamic acid at Kabat position 1, valine at Kabat position 24, isoleucine at Kabat position 37, lysine at Kabat position 43, arginine at Kabat position 44, methionine at Kabat position 48, serine at Kabat position 70, leucine at Kabat position 80, glutamic acid at Kabat position 81, tryptophan at Kabat position 82, alanine at Kabat position 84, serine at Kabat position 85, or valine or methionine at Kabat position 89.

In some embodiments, the humanized light chain variable region contains glutamic acid or alanine at the Kabat position 1, valine

- 27 042795 at Kabat position 2, glutamine at Kabat position 3, methionine at Kabat position 4, aspartic acid or leucine at Kabat position 9, phenylalanine or threonine at position Kabat position 10, glutamine at Kabat position 11, serine or proline at Kabat position 12, alanine or leucine at Kabat position 13, threonine at Kabat position 14 , valine or proline at Kabat position 15, lysine at Kabat position 16, glutamic acid or aspartic acid at Kabat position 17, lysine or proline at Kabat position 18, threonine at Kabat position 22, lysine at Kabat position 42, arginine at Kabat position 45, isoleucine at Kabat position 58, proline or serine at Kabat position 60 , tyrosine at Kabat position 67, phenylalanine at Kabat position 73, isoleucine at Kabat position 78, serine or alanine at Kabat position 80, tyrosine at position 85 according to Kabat, and phenylalanine in the position corresponding to position 87 according to Kabat; and the humanized heavy chain variable region contains glutamic acid at Kabat position 1, valine at Kabat position 24, isoleucine at Kabat position 37, lysine at Kabat position 43, arginine at Kabat position 44, methionine at Kabat position 48, serine at Kabat position 70, leucine at Kabat position 80, glutamic acid at Kabat position 81, tryptophan at Kabat position 82, alanine at Kabat position 84, serine at Kabat position 85, or valine or methionine at Kabat position 89.

In some embodiments, the humanized light chain variable region comprises glutamic acid or alanine at Kabat position 1, valine at Kabat position 2, glutamine at Kabat position 3, methionine at position 4 Kabat, aspartic acid or leucine at Kabat position 9, phenylalanine or threonine at Kabat position 10, glutamine at Kabat position 11, serine or proline at Kabat position 12, alanine or leucine at Kabat position 13, threonine at Kabat position 14, valine or proline at Kabat position 15, lysine at Kabat position 16, glutamic acid or aspartic acid at Kabat position 17, lysine or proline in Kabat position 18, threonine in Kabat position 22, lysine in Kabat position 42, arginine in Kabat position 45, isoleucine at Kabat position 58, proline or serine at Kabat position 60, tyrosine at Kabat position 67, phenylalanine at Kabat position 73, isoleucine at Kabat position 78 Kabat, serine or alanine at Kabat position 80, tyrosine at Kabat position 85, or phenylalanine at Kabat position 87; and the humanized heavy chain variable region contains glutamic acid at Kabat position 1, valine at Kabat position 24, isoleucine at Kabat position 37, lysine at Kabat position 43, arginine at Kabat position 44, methionine at Kabat position 48, serine at Kabat position 70, leucine at Kabat position 80, glutamic acid at Kabat position 81, tryptophan at Kabat position 82, alanine at Kabat position 84, serine at Kabat position 85, and valine or methionine at Kabat position 89.

In some embodiments, the humanized light chain variable region comprises glutamic acid or alanine at Kabat position 1, valine at Kabat position 2, glutamine at Kabat position 3, methionine at position 4 Kabat, aspartic acid or leucine at Kabat position 9, phenylalanine or threonine at Kabat position 10, glutamine at position

- 28 042795 Kabat 11, serine or proline at Kabat position 12, alanine or leucine at Kabat position 13, threonine at Kabat position 14, valine or proline at position 15 Kabat, lysine at position 16 Kabat, glutamic acid or aspartic acid at position 17 Kabat, lysine or proline at position 18 Kabat, threonine at position 22 Kabat, lysine at Kabat position 42, arginine at Kabat position 45, isoleucine at Kabat position 58, proline or serine at Kabat position 60, tyrosine at Kabat position 67 Kabat, phenylalanine at Kabat position 73, isoleucine at Kabat position 78, serine or alanine at Kabat position 80, tyrosine at Kabat position 85, and phenylalanine at Kabat position position 87 of Kabat; and the humanized heavy chain variable region contains glutamic acid at Kabat position 1, valine at Kabat position 24, isoleucine at Kabat position 37, lysine at Kabat position 43, arginine at Kabat position 44, methionine at Kabat position 48, serine at Kabat position 70, leucine at Kabat position 80, glutamic acid at Kabat position 81, tryptophan at Kabat position 82, alanine at Kabat position 84, serine at Kabat position 85, and valine or methionine at Kabat position 89.

In some embodiments, the humanized heavy chain variable region comprises valine at Kabat position 5, serine at Kabat position 7, valine at Kabat position 11, glutamic acid at Kabat position 12 , valine at Kabat position 20, arginine at Kabat position 38, alanine at Kabat position 40, methionine at Kabat position 48, arginine at Kabat position 66 , valine at Kabat position 67, isoleucine at Kabat position 69, alanine at Kabat position 71, lysine at Kabat position 73, threonine at Kabat position 75 , glutamic acid at Kabat position 81, arginine at Kabat position 83, threonine at Kabat position 87, or valine at Kabat position 89.

In some embodiments, the humanized heavy chain variable region comprises valine at Kabat position 5, serine at Kabat position 7, valine at Kabat position 11, glutamic acid at Kabat position 12 , valine at Kabat position 20, arginine at Kabat position 38, alanine at Kabat position 40, methionine at Kabat position 48, arginine at Kabat position 66 , valine at Kabat position 67, isoleucine at Kabat position 69, alanine at Kabat position 71, lysine at Kabat position 73, threonine at Kabat position 75 , glutamic acid at Kabat position 81, arginine at Kabat position 83, threonine at Kabat position 87, and valine at Kabat position 89.

In some embodiments, the humanized heavy chain variable region comprises the sequence from SEQ ID NO: 7. In some embodiments, the humanized heavy chain variable region is SEQ ID NO: 7.

In some embodiments, the humanized heavy chain variable region comprises the sequence from SEQ ID NO: 53. In some embodiments, the humanized heavy chain variable region is SEQ ID NO: 53. In some embodiments, the humanized light chain variable region comprises the sequence from SEQ ID NO: : 55. In some embodiments, the humanized light chain variable region is SEQ ID NO: 55. Thus, another aspect relates to a humanized 1E9 antibody comprising a humanized light chain variable region and a humanized heavy chain variable region, wherein the humanized heavy chain variable region contains the sequence from SEQ ID NO: 53, and the humanized light chain variable region contains the sequence from SEQ ID NO: 55.

- 29 042795

In addition, the present invention relates to humanized 1E9 antibodies capable of binding CD73 and comprising a humanized light chain variable region and a humanized heavy chain variable region, including the sequence from SEQ ID NO: 7. Thus, another aspect relates to a humanized 1E9 antibody comprising the variable region a humanized light chain region; and a humanized heavy chain variable region, wherein the humanized heavy chain variable region contains the sequence from SEQ ID NO: 7.

Humanized 1E9 antibodies as described herein may be Fab' fragments. When the humanized 1E9 antibodies are Fab' fragments, the humanized 1E9 antibodies include a humanized heavy chain (eg, including a constant and variable region) and a humanized light chain (eg, including a constant and variable region). In some embodiments, the humanized 1E9 antibody is a Fab' fragment. In some embodiments, the humanized 1E9 antibody comprises a human constant region. In some embodiments, the humanized 1E9 antibody is an IgG. In some embodiments, the humanized 1E9 antibody is IgG1. In some embodiments, the humanized 1E9 antibody is IgG4. In some embodiments, the humanized 1E9 antibody is IgA. In other embodiments, the humanized antibody is an IgM.

In some embodiments, the humanized 1E9 antibody is a single chain antibody. A single chain antibody includes a variable light chain and a variable heavy chain. One of skill in the art will immediately recognize that a single chain antibody comprises one light chain and one heavy chain, in contrast to an immunoglobulin antibody comprising two identical pairs of polypeptide chains, where each pair has one light chain and one heavy chain. Each light chain and heavy chain, in turn, consists of two regions: a variable (V) region (i.e., variable light chain and variable heavy chain) involved in target antigen binding, and a constant (C) region interacting with other components of the immune system. The variable light chain and the variable heavy chain in a single chain antibody may be linked via a linker peptide. Examples of linker peptides from single chain antibodies are described in Bird, R. E., Hardman, K. D., Jacobson, J. W., Johnson, S., Kaufman, B. M., Lee, S. M., Lee, T., Pope, S. H., Riordan, G. S. and Whitlow, M. (1988). Methods for producing scFv antibodies are described. See, Huse et al., Science 246:12751281 (1989); Ward et al., Nature 341:544-546 (1989); and Vaughan et al., Nature Biotech. 14:309-314 (1996). Briefly, mRNA is isolated from B cells from an immunized animal and cDNA is obtained. cDNA is amplified using primers specific for the variable regions of immunoglobulin heavy and light chains. The PCR products are purified and the nucleic acid sequences are joined. If a linker peptide is desired, nucleic acid sequences encoding the peptide are inserted between the heavy and light chain nucleic acid sequences. The nucleic acid encoding the scFv is inserted into a vector and expressed in a suitable host cell.

The ability of an antibody to bind a specific epitope (eg, CD73) can be described by the equilibrium dissociation constant (KD). The equilibrium dissociation constant (KD), as defined herein, is the ratio of the rate of dissociation (K _off ) and the rate of binding (K _on ) of a humanized 1E9 antibody to the CD73 protein. It is described by the following formula:

^K D= ^K off/ ^K on.

In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (KD) of about 0.5 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (KD) of about 1 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (KD) of about 1.5 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (K _D ) of about 2 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (K _D ) of about 2.5 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (KD) of about 3 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (KD) of about 3.5 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (KD) of about 4 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH below 7.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen to

- 30 042795 equilibrium dissociation constant (K _D ) in this paragraph at a pH of less than about 7.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of less than about 7.0. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of less than about 6.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of less than about 6.0. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 5.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K D ) in this paragraph at a pH of less than about 5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at pH less than about 4.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of about 6.0 to about 7.0. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.0. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.1. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.2. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.3. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.4. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.6. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.7. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.8. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.9. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of approximately 7.0.

In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (K _D ) of about 4.5 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (K _D ) of about 5 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (K _D ) of about 5.5 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (KD) of about 6 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (K _D ) of about 6.5 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (K _D ) of about 7 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (K _D ) of about 7.5 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (K _D ) of about 8 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH below 7.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of less than about 7.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 7.0. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of less than about 6.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen to

- 31 042795 equilibrium dissociation constant (K _D ) in this paragraph at a pH of less than about 6.0. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of less than about 5.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH less than about 5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH less than about 4.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of about 6.0 to about 7.0. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of approximately 6.0. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.1. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.2. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.3. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.4. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of approximately 6.6. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of approximately 6.7. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.8. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.9. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 7.0.

In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (K _D ) of about 8.5 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (KD) of about 9 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (K _D ) of about 9.5 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (K _D ) of about 10 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (K _D ) of about 11 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (KD) of about 12 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (KD) of about 13 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (KD) of about 14 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (K _D ) of about 15 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (K _D ) of about 16 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH below 7.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 7.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 7.0. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 6.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of less than about 6.0. In some embodiments, the humanized antibody is capable of binding the CD73 antigen to

- 32 042795 equilibrium dissociation constant (K _D ) in this paragraph at a pH of less than about 5.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH less than about 5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH less than about 4.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of about 6.0 to about 7.0. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of approximately 6.0. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.1. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.2. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.3. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.4. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of approximately 6.6. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of approximately 6.7. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.8. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.9. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 7.0.

In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (K _D ) of about 17 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (KD) of about 18 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (KD) of about 19 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (KD) of about 20 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (K _D ) of about 21 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (K _D ) of about 22 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (KD) of about 23 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (KD) of about 24 to about 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (K _D ) of approximately 0.5.12.3.4.5.6.7.8.9.10.11.12.13.14 , 15, 16, 17.18, 19, 20, 21, 22, 23, 24, or 25 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH below 7.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of less than about 7.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of less than about 7.0. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 6.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 6.0. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 5.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with the equilibrium dissociation constant (K _D ) in this paragraph at a pH less than about 5. In some embodiments, the humanized

- 33 042795 the antibody is capable of binding the CD73 antigen with the equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 4.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.0 to about 7.0. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of approximately 6.0. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of approximately 6.1. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of approximately 6.2. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of approximately 6.3. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of approximately 6.4. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of approximately 6.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of approximately 6.6. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of approximately 6.7. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of approximately 6.8. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of approximately 6.9. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of approximately 7.0.

In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (KD) of approximately 7.1 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (KD) of approximately 6.9 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (KD) of approximately 9.4 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (KD) of approximately 19.5 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (KD) of approximately 17.8 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (KD) of approximately 15.9 nM. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH below 7.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 7.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 7.0. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 6.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 6.0. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 5.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at pH less than about 4.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.0 to about 7.0. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of approximately 6.0. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of approximately 6.1. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of approximately 6.2. In some embodiments, the humanized antibody is capable of binding the CD73 antigen to an equilibrium constant

- 34 042795 of that dissociation (K _D ) in this paragraph at a pH of approximately 6.3. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.4. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.6. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.7. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (K _D ) in this paragraph at a pH of approximately 6.8. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of approximately 6.9. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at the equilibrium dissociation constant (KD) in this paragraph at a pH of approximately 7.0.

In one aspect, the present invention provides an antibody capable of binding CD73 at a pH of less than about 7.5. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH less than about 7.0. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH less than about 6.5. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH less than about 6.0. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH less than about 5.5. In some embodiments, the antibody is capable of binding to the CD73 antigen at a pH of less than about 5. In some embodiments, the antibody is capable of binding to the CD73 antigen at a pH of less than about 4.5. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH of about 6.0 to about 7.0. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH of about 6.0. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH of about 6.1. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH of about 6.2. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH of about 6.3. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH of about 6.4. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH of about 6.5. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH of about 6.6. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH of about 6.7. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH of about 6.8. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH of about 6.9. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH of approximately 7.0. In some embodiments, the implementation of the antibody, as indicated in this paragraph, is a humanized antibody. In some embodiments, the antibody comprises a light chain variable region (eg, a humanized light chain) and a heavy chain variable region (eg, a humanized heavy chain). The variable region of the light chain contains:

(i) L1 CDR (e.g. mouse CDR L1) as set forth in SEQ ID NO: 1, L2 CDR (e.g. mouse CDR L2) as set out in SEQ ID NO: 2, L3 CDR (e.g. mouse CDR L3) as indicated in SEQ ID NO: 3, and (ii) valine at position corresponding to position 2 according to Kabat, methionine at position corresponding to position 4 according to Kabat, aspartic acid or leucine at position corresponding to position 9 according to Kabat, proline or serine at Kabat position 12, lysine or proline at Kabat position 18, alanine at Kabat position 43, proline or serine at Kabat position 60, threonine at position 74 Kabat, asparagine or serine at Kabat position 76, asparagine or serine at Kabat position 77, isoleucine or leucine at Kabat position 78, serine or alanine at Kabat position 80 , glutamine at Kabat position 100, valine at Kabat position 104, glutamic acid or alanine at Kabat position 1, glutamine at Kabat position 3, phenylalanine or threonine at position Kabat position 10, glutamine at Kabat position 11, alanine or leucine at Kabat position 13, threonine at Kabat position 14, valine or proline at Kabat position 15, lysine in position corresponding to position 16 according to Kabat, glutamic acid or aspartic acid in position corresponding to position 17 according to Kabat, threonine in position,

- 35 042795 Kabat position 22, lysine at Kabat position 42, arginine at Kabat position 45, isoleucine at Kabat position 58, tyrosine at Kabat position 67, phenylalanine at Kabat position 73, isoleucine at Kabat position 78, tyrosine at Kabat position 85, or phenylalanine at Kabat position 87.

The heavy chain variable region contains:

(i) CDR H1 (e.g. mouse CDR H1) as set forth in SEQ ID NO: 4, CDRH2 (e.g. mouse CDR H2) as listed in SEQ ID NO: 5, CDR H3 (e.g. mouse CDR H3), as indicated in SEQ ID NO: 6, and (ii) isoleucine at position corresponding to position 37 Kabat, alanine or proline at position corresponding to position 40 Kabat, lysine at position corresponding to position 43 Kabat, serine at position corresponding Kabat position 70, isoleucine or threonine at Kabat position 75, tryptophan at Kabat position 82, arginine or lysine at Kabat position 83, alanine at Kabat position 84, serine at Kabat position 85, valine or methionine at Kabat position 89, valine at Kabat position 5, serine at Kabat position 7, valine at Kabat position 11 , glutamic acid or lysine at Kabat position 12, isoleucine or valine at Kabat position 20, arginine at Kabat position 38, arginine at Kabat position 66, valine at Kabat position 67, isoleucine at Kabat position 69, alanine at Kabat position 71, lysine at Kabat position 73, threonine at Kabat position 87, glutamic acid at position , corresponding to Kabat position 1, valine at position corresponding to Kabat position 24, arginine at position corresponding to Kabat position 44, methionine at position corresponding to Kabat position 48, leucine at position corresponding to Kabat position 80, or glutamic acid in a position corresponding to position 81 according to Kabat.

The humanized 1E9 antibodies provided herein are capable of binding CD73 at a pH below 7.5. Thus, in some embodiments, the humanized antibody is capable of binding the CD73 antigen at a pH of less than about 7.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at a pH less than about 7.0. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at a pH less than about 6.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at a pH less than about 6.0. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at a pH less than about 5.5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at a pH of less than about 5. In some embodiments, the humanized antibody is capable of binding the CD73 antigen at a pH of less than about 4.5. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH of about 6.0 to about 7.0. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH of about 6.0. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH of about 6.1. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH of about 6.2. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH of about 6.3. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH of about 6.4. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH of about 6.5. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH of about 6.6. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH of about 6.7. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH of about 6.8. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH of about 6.9. In some embodiments, the antibody is capable of binding the CD73 antigen at a pH of approximately 7.0.

The humanized 1E9 antibody provided herein, including embodiments thereof, may include glutamine at a position corresponding to Kabat position 297.

In some embodiments, the humanized 1E9 antibody is linked to the CD73 antigen. In some embodiments, the CD73 antigen forms part of the cell. In some embodiments, the cell is a lymphoid cell. In some embodiments, the cell is a T cell. In some embodiments, the implementation of the cell is a malignant cell.

- 36 042795

In one aspect, the present invention provides a humanized 1E9 antibody bound to the CD73 antigen at a pH of less than about 7.5. In some embodiments, the humanized 1E9 antibody comprises a humanized light chain variable region and a humanized heavy chain variable region, wherein the humanized light chain variable region comprises isoleucine at Kabat position 2, leucine at Kabat position 4, serine, or alanine in Kabat position 9, serine or threonine in Kabat position 10, leucine in Kabat position 11, serine in Kabat position 14, glycine in Kabat position 16 , arginine at Kabat position 18, threonine at Kabat position 20, or glutamine at Kabat position 42; and wherein the humanized heavy chain variable region comprises glutamine at Kabat position 1, valine or glutamic acid at Kabat position 12, serine at Kabat position 17, methionine or valine at position 20 Kabat, alanine at Kabat position 24, valine at Kabat position 37, arginine or alanine at Kabat position 40, proline at Kabat position 41, glutamine at Kabat position Kabat position 43, glycine at Kabat position 44, threonine at Kabat position 70, threonine at Kabat position 75, methionine at Kabat position 80, threonine or arginine at position , corresponding to position 83 according to Kabat, serine in position corresponding to position 84 according to Kabat, glutamic acid in position corresponding to position 85 according to Kabat, or valine in position corresponding to position 89 according to Kabat.

In some embodiments, the humanized light chain variable region comprises isoleucine at Kabat position 2, leucine at Kabat position 4, serine or threonine at Kabat position 10, leucine at Kabat position 11 Kabat, threonine at Kabat position 20 and glutamine at Kabat position 42; and the humanized heavy chain variable region contains glutamine at Kabat position 1, serine at Kabat position 17, methionine or valine at Kabat position 20, alanine at Kabat position 24, valine at Kabat position 37, arginine or alanine at Kabat position 40, proline at Kabat position 41, glutamine at Kabat position 43, glycine at Kabat position 44 , threonine at Kabat position 70, threonine at Kabat position 75, methionine at Kabat position 80, threonine or arginine at Kabat position 83, serine at position 84 according to Kabat, glutamic acid at position corresponding to position 85 according to Kabat, and valine at position corresponding to position 89 according to Kabat.

In some embodiments, the humanized light chain variable region comprises isoleucine at Kabat position 2, leucine at Kabat position 4, serine or threonine at Kabat position 10, leucine at Kabat position 11 Kabat, threonine at Kabat position 20 or glutamine at Kabat position 42; and the humanized heavy chain variable region contains glutamine at Kabat position 1, serine at Kabat position 17, methionine or valine at Kabat position 20, alanine at Kabat position 24, valine at Kabat position 37, arginine or alanine at Kabat position 40, proline at Kabat position 41, glutamine at Kabat position 43, glycine at Kabat position 44 , threonine at Kabat position 70, threonine at Kabat position 75, methionine at Kabat position 80, threonine or arginine at Kabat position 83, serine at position 84 according to Kabat, glutamic acid in position corresponding to position 85 according to Kabat or valine in position corresponding to position 89 according to Kabat.

In some embodiments, the humanized light chain variable region comprises isoleucine at Kabat position 2, leucine at Kabat position 4, serine or threonine at Kabat position 10, leucine at Kabat position 11 Kabat, threonine at Kabat position 20 and glutamine at Kabat position 42; And

The humanized heavy chain variable region contains glutamine at position corresponding to Kabat position 1, serine at position corresponding to Kabat position 17, methionine or valine at position corresponding to Kabat position 20, alanine at position corresponding to Kabat position 24 , valine at Kabat position 37, arginine or alanine at Kabat position 40, proline at Kabat position 41, glutamine at Kabat position 43, glycine at position 44 Kabat, threonine at Kabat position 70, threonine at Kabat position 75, methionine at Kabat position 80, threonine or arginine at Kabat position 83, serine at position Kabat position 84, glutamic acid at Kabat position 85, or valine at Kabat position 89.

In some embodiments, the humanized light chain variable region comprises isoleucine at Kabat position 2, leucine at Kabat position 4, serine or threonine at Kabat position 10, leucine at Kabat position 11 Kabat, threonine at Kabat position 20 or glutamine at Kabat position 42; and the humanized heavy chain variable region contains glutamine at Kabat position 1, serine at Kabat position 17, methionine or valine at Kabat position 20, alanine at Kabat position 24, valine at Kabat position 37, arginine or alanine at Kabat position 40, proline at Kabat position 41, glutamine at Kabat position 43, glycine at Kabat position 44 , threonine at Kabat position 70, threonine at Kabat position 75, methionine at Kabat position 80, threonine or arginine at Kabat position 83, serine at position 84 according to Kabat, glutamic acid at position corresponding to position 85 according to Kabat, and valine at position corresponding to position 89 according to Kabat.

In some embodiments, the humanized light chain variable region comprises serine or alanine at Kabat position 9, serine at Kabat position 14, glycine at Kabat position 16, and arginine at Kabat position 18. Kabat; and the variable region of the humanized heavy chain contains valine or glutamic acid at a position corresponding to position 12 according to Kabat.

In some embodiments, the humanized light chain variable region comprises serine or alanine at position corresponding to Kabat position 9, serine at position corresponding to Kabat position 14, glycine at position corresponding to Kabat position 16, or arginine at position corresponding to position 18 by Kabat; and the variable region of the humanized heavy chain contains valine or glutamic acid at a position corresponding to position 12 according to Kabat.

In some embodiments, the pH is from about 6.0 to about 7.0. In some embodiments, the pH is about 6.7. In some embodiments, the pH is about 6.3. In some embodiments, the antibody inhibits the catalytic activity of said CD73 antigen. In some embodiments, the antibody comprises a humanized light chain variable region, including the sequence from SEQ ID NO: 36 or SEQ ID NO: 37. In some embodiments, the antibody comprises a humanized heavy chain variable region, including the sequence from SEQ ID NO: 7. In some embodiments, embodiments, the CD73 antigen forms part of the cell. In some embodiments, the CD73 antigen is associated with a solid support.

In another aspect, the present invention provides an anti-CD73 antibody. The anti-CD73 antibody binds the same epitope as the 1E9 antibody, wherein the 1E9 antibody contains a humanized light chain variable region, including mouse CDR L1, mouse CDR L2, or mouse CDR L3, and a humanized heavy chain variable region, including mouse CDR H1, CDR H2 mouse or CDR H3 mouse.

In another aspect, the present invention provides an anti-CD73 antibody. The CD73 antibody binds the same epitope as the 1E9 antibody, wherein the 1E9 antibody comprises a humanized light chain variable region and a humanized heavy chain variable region. The variable region of the humanized light chain contains:

(i) mouse CDR L1 as listed in SEQ ID NO: 1, mouse CDR L2 as listed in SEQ ID NO: 2, mouse CDR L3 as listed in SEQ ID NO: 3, and (ii) valine at the position, corresponding to position 2 according to Kabat, methionine in position corresponding to

- 38 042795 current Kabat position 4, aspartic acid or leucine in position corresponding to position 9 in Kabat, proline or serine in position corresponding to position 12 in Kabat, lysine or proline in position corresponding to position 18 in Kabat, alanine in position, Kabat position 43, proline or serine at Kabat position 60, threonine at Kabat position 74, asparagine or serine at Kabat position 76, asparagine or serine at Kabat position 77 Kabat, isoleucine or leucine at Kabat position 78, serine or alanine at Kabat position 80, glutamine at Kabat position 100, valine at Kabat position 104, glutamic acid or alanine in Kabat position 1, glutamine in Kabat position 3, phenylalanine or threonine in Kabat position 10, glutamine in Kabat position 11, alanine or leucine in position 13 Kabat, threonine at Kabat position 14, valine or proline at Kabat position 15, lysine at Kabat position 16, glutamic acid or aspartic acid at Kabat position 17, threonine at Kabat position 22, lysine at Kabat position 42, arginine at Kabat position 45, isoleucine at Kabat position 58, tyrosine at Kabat position 67, phenylalanine at Kabat position 73, isoleucine at Kabat position 78, tyrosine at Kabat position 85, or phenylalanine at Kabat position 87.

The variable region of a humanized heavy chain contains:

(i) mouse H1 CDR as set out in SEQ ID NO: 4, mouse H2 CDR as set out in SEQ ID NO: 5, and mouse H3 CDR as set out in SEQ ID NO: 6, and (ii) isoleucine at position Kabat position 37, alanine or proline at Kabat position 40, lysine at Kabat position 43, serine at Kabat position 70, isoleucine or threonine at Kabat position 75 , tryptophan at Kabat position 82, arginine or lysine at Kabat position 83, alanine at Kabat position 84, serine at Kabat position 85, valine or methionine at position Kabat position 89, valine at Kabat position 5, serine at Kabat position 7, valine at Kabat position 11, glutamic acid or lysine at Kabat position 12, isoleucine or valine at Kabat position 20, arginine at Kabat position 38, arginine at Kabat position 66, valine at Kabat position 67, isoleucine at Kabat position 69, alanine at Kabat position 71, lysine at Kabat position 73, threonine at Kabat position 87, glutamic acid at Kabat position 1, valine at Kabat position 24 , arginine at Kabat position 44, methionine at Kabat position 48, leucine at Kabat position 80, or glutamic acid at Kabat position 81.

Humanized IgG1 antibodies

One aspect of the present disclosure relates to a humanized IgG1 antibody comprising a humanized light chain variable region and a humanized heavy chain variable region, wherein the humanized light chain variable region comprises a murine CDR L1 as set forth in SEQ ID NO: 1, a murine CDR L2 as set forth in SEQ ID NO: 2, mouse CDR L3 as set forth in SEQ ID NO: 3, and wherein the humanized heavy chain variable region contains mouse CDR H1 as set forth in SEQ ID NO: 4, mouse CDR H2 as set forth in SEQ ID NO : 5, and mouse H3 CDRs as indicated in SEQ ID NO: 6.

One aspect of the present disclosure relates to a humanized IgG4 antibody comprising a humanized light chain variable region and a humanized heavy chain variable region, wherein the humanized light chain variable region comprises a murine CDR L1 as set forth in SEQ ID NO: 1, a murine CDR L2 as set forth in SEQ ID NO: 2, mouse CDR L3 as set forth in SEQ ID NO: 3, and wherein the humanized heavy chain variable region contains mouse CDR H1 as set forth in SEQ ID NO: 4, mouse CDR H2 as set forth in SEQ ID NO : 5, and mouse H3 CDRs as indicated in SEQ ID NO: 6.

The humanized light chain variable region may include a mouse CDR L1 as set forth in SEQ ID NO: 1, a mouse CDR L2 as set forth in SEQ ID NO: 2, or a mouse CDR L3 as set forth in SEQ ID NO: 3. The light chain may include a mouse L1 CDR as set forth in SEQ ID NO: 1, a mouse L2 CDR as set forth in SEQ ID NO: 2, and a mouse L3 CDR as set forth in SEQ ID NO: 3. A humanized heavy chain variable region may enable cdr h1 mouse like uka

- 39 042795 is specified in SEQ ID NO: 4, a mouse H2 CDR as specified in SEQ ID NO: 5, or a mouse H3 CDR as specified in SEQ ID NO: 6. A humanized heavy chain variable region may include a mouse H1 CDR as indicated in SEQ ID NO: 4, a mouse H2 CDR as indicated in SEQ ID NO: 5, and a mouse H3 CDR as indicated in SEQ ID NO: 6. In some embodiments, the humanized light chain variable region comprises a mouse CDR L1, as set forth in SEQ ID NO: 1. In some embodiments, the humanized light chain variable region comprises a mouse L2 CDR as set forth in SEQ ID NO: 2. In some embodiments, the humanized light chain variable region comprises a mouse L3 CDR as set forth in SEQ ID NO: 3. In some embodiments, the humanized heavy chain variable region comprises a mouse H1 CDR as set forth in SEQ ID NO: 4. In some embodiments, the humanized heavy chain variable region comprises a mouse H2 CDR as set forth in SEQ ID NO: 5 In some embodiments, the humanized light chain variable region comprises a mouse H3 CDR as indicated in SEQ ID NO: 6.

In some embodiments, the humanized light chain variable region further comprises a valine at Kabat position 2, a methionine at Kabat position 4, aspartic acid or leucine at Kabat position 9, a proline or serine at Kabat position 12, lysine or proline at Kabat position 18, alanine at Kabat position 43, proline or serine at Kabat position 60, threonine at Kabat position 74, asparagine or serine at Kabat position 76, asparagine or serine at Kabat position 77, isoleucine or leucine at Kabat position 78, serine or alanine at Kabat position 80, glutamine at Kabat position 100, valine at Kabat position 104, glutamic acid or alanine at Kabat position 1, glutamine at Kabat position 3, phenylalanine or threonine at position 10 Kabat, glutamine at Kabat position 11, alanine or leucine at Kabat position 13, threonine at Kabat position 14, valine or proline at Kabat position 15, lysine at position Kabat position 16, glutamic acid or aspartic acid at Kabat position 17, threonine at Kabat position 22, lysine at Kabat position 42, arginine at Kabat position 45 , isoleucine at Kabat position 58, tyrosine at Kabat position 67, phenylalanine at Kabat position 73, isoleucine at Kabat position 78, tyrosine at Kabat position 85 or phenylalanine at the position corresponding to position 87 according to Kabat.

In some embodiments, the humanized light chain variable region further comprises a valine at Kabat position 2, a methionine at Kabat position 4, aspartic acid or leucine at Kabat position 9, a proline or serine at Kabat position 12, lysine or proline at Kabat position 18, alanine at Kabat position 4 3, proline or serine at Kabat position 60, threonine at Kabat position 74 , asparagine or serine at Kabat position 76, asparagine or serine at Kabat position 77, isoleucine or leucine at Kabat position 78, serine or alanine at Kabat position 80, glutamine at Kabat position 100, valine at Kabat position 104, glutamic acid or alanine at Kabat position 1, glutamine at Kabat position 3, phenylalanine or threonine at position Kabat position 10, glutamine at Kabat position 11, alanine or leucine at Kabat position 13, threonine at Kabat position 14, valine or proline at Kabat position 15, lysine at Kabat position 16, glutamic acid or aspartic acid in Kabat position 17, threonine in Kabat position 22, lysine in Kabat position 4, arginine in position 4 5 Kabat, isoleucine at position 58 Kabat, tyrosine at position 67 Kabat, phenylalanine at position 73 Kabat, isoleucine at position 78 Kabat, tyrosine at position 85 according to Kabat and phenylalanine in the position corresponding to position 87 according to Kabat.

In some embodiments, the humanized heavy chain variable region further comprises isoleucine at position corresponding to Kabat position 37, alanine, or

- 40 042795 Proline at Kabat position 40, Lysine at Kabat position 43, Serine at Kabat position 70, Isoleucine or Threonine at Kabat position 75, Tryptophan at Kabat position Kabat position 82, arginine or lysine at Kabat position 83, alanine at Kabat position 84, serine at Kabat position 85, valine or methionine at Kabat position 89, valine at Kabat position 5, serine at Kabat position 7, valine at Kabat position 11, glutamic acid or lysine at Kabat position 12, isoleucine or valine at position Kabat 20, arginine at Kabat 38, arginine at Kabat 66, valine at Kabat 67, isoleucine at Kabat 69, alanine at Kabat 71 Kabat, lysine at position 73 Kabat, threonine at position 87 Kabat, glutamic acid at position 1 Kabat, valine at position 24 Kabat, arginine at position Kabat position 44, methionine at Kabat position 48, leucine at Kabat position 80, or glutamic acid at Kabat position 81.

In some embodiments, the humanized heavy chain variable region further comprises isoleucine at Kabat position 37, alanine or proline at Kabat position 40, lysine at Kabat position 43, serine at position 70 Kabat, isoleucine or threonine at Kabat position 75, tryptophan at Kabat position 82, arginine or lysine at Kabat position 83, alanine at Kabat position 84, serine at position , corresponding to Kabat position 85, valine or methionine in position corresponding to Kabat position 89, valine in position corresponding to Kabat position 5, serine in position corresponding to Kabat position 7, valine in position corresponding to Kabat position 11, glutamine acid or lysine at Kabat position 12, isoleucine or valine at Kabat position 20, arginine at Kabat position 38, arginine at Kabat position 66, valine at Kabat position 67 Kabat, isoleucine at position 69 Kabat, alanine at position 71 Kabat, lysine at position 73 Kabat, threonine at position 87 Kabat, glutamic acid at position Kabat position 1, valine at Kabat position 24, arginine at Kabat position 44, methionine at Kabat position 48, leucine at Kabat position 80, and glutamic acid at position compliant with regulation 81 of Kabat.

In some embodiments, the humanized light chain variable region further comprises valine at Kabat position 2, methionine at Kabat position 4, leucine at Kabat position 9, proline at Kabat position 12 , and proline at the position corresponding to position 18 according to Kabat.

In some embodiments, the humanized heavy chain variable region further comprises isoleucine at Kabat position 37, proline at Kabat position 40, lysine at Kabat position 43, serine at Kabat position 70 , isoleucine at Kabat position 75, tryptophan at Kabat position 82, lysine at Kabat position 83, alanine at Kabat position 84, serine at Kabat position 85 , and methionine at the position corresponding to position 89 according to Kabat.

In some embodiments, the humanized heavy chain variable region comprises valine at Kabat position 5, serine at Kabat position 7, valine at Kabat position 11, glutamic acid at Kabat position 12 , valine at Kabat position 20, arginine at Kabat position 38, alanine at Kabat position 40, methionine at Kabat position 48, arginine at Kabat position 66 , valine at Kabat position 67, isoleucine at Kabat position 69, alanine at Kabat position 71, lysine at

- 41 042795 Kabat position 73, threonine at Kabat position 75, glutamic acid at Kabat position 81, arginine at Kabat position 83, threonine at Kabat position 87, or valine in a position corresponding to position 89 according to Kabat.

In some embodiments, the humanized heavy chain variable region comprises valine at Kabat position 5, serine at Kabat position 7, valine at Kabat position 11, glutamic acid at Kabat position 12 , valine at Kabat position 20, arginine at Kabat position 38, alanine at Kabat position 40, methionine at Kabat position 48, arginine at Kabat position 66 , valine at Kabat position 67, isoleucine at Kabat position 69, alanine at Kabat position 71, lysine at Kabat position 73, threonine at Kabat position 75 , glutamic acid at Kabat position 81, arginine at Kabat position 83, threonine at Kabat position 87, and valine at Kabat position 89.

In some embodiments, the humanized IgG1 antibody further comprises glutamine at a position corresponding to Kabat position 297. In some embodiments, the humanized IgG1 antibody provided herein, including embodiments thereof, is associated with the CD73 antigen. In some embodiments, the CD73 antigen forms part of the cell. In some embodiments, the cell is a T cell. In some embodiments, the implementation of the cell is a malignant cell.

Nucleic acid compositions

In one aspect, the present invention provides an isolated nucleic acid encoding a humanized 1E9 antibody as described herein, including embodiments thereof. The humanized 1E9 antibody encoded by the isolated nucleic acid is described in detail throughout this specification (including the description above and the examples section). Thus, a humanized antibody encoded by an isolated nucleic acid includes all of the embodiments described herein. For example, the nucleic acid may encode at least one CDR, specific residues involved in epitope binding, or binding framework region residues. For example, the nucleic acid may encode a humanized light chain including a valine at a position corresponding to Kabat position 2.

In some embodiments, the isolated nucleic acid comprises a sequence from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. In some embodiments, the isolated nucleic acid comprises a sequence from SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27. In some embodiments, the isolated nucleic acid comprises a sequence of SEQ ID NO: 8 and the sequence from SEQ ID NO: 18. In some embodiments, the isolated nucleic acid contains the sequence from SEQ ID NO: 9 and the sequence from SEQ ID NO: 19. In some embodiments, the isolated nucleic acid contains the sequence from SEQ ID NO: 19. NO: 10 and the sequence from SEQ ID NO: 20. In some embodiments, the isolated nucleic acid contains the sequence from SEQ ID NO: 11 and the sequence from SEQ ID NO: 21. In some embodiments, the isolated nucleic acid contains the sequence from SEQ ID NO: 12 and the sequence from SEQ ID NO: 22. In some embodiments, the isolated nucleic acid contains the sequence from SEQ ID NO: 13 and the sequence from SEQ ID NO: 23. In some embodiments, the isolated nucleic acid contains the sequence from SEQ ID NO: 14 and sequence from SEQ ID NO: 24. In some embodiments, the isolated nucleic acid contains the sequence from SEQ ID NO: 15 and the sequence from SEQ ID NO: 25. In some embodiments, the isolated nucleic acid contains the sequence from SEQ ID NO: 16 and the sequence from SEQ ID NO: 26. In some embodiments, the isolated nucleic acid comprises the sequence from SEQ ID NO: 17 and the sequence from SEQ ID NO: 27.

In some embodiments, the isolated nucleic acid comprises a codon-optimized sequence. In some embodiments, the isolated nucleic acid comprises SEQ ID NO: 52 or SEQ ID NO: 54. In some embodiments, the isolated nucleic acid comprises SEQ ID NO: 52. In some embodiments, the isolated nucleic acid comprises SEQ ID NO: 54. In some embodiments, the isolated nucleic acid comprises SEQ ID NO: 54. embodiments, the isolated nucleic acid is SEQ ID NO: 52 or SEQ ID NO: 54. In some embodiments, the isolated nucleic acid is SEQ ID NO: 52. In some embodiments,

- 42 042795 An embodiment of the isolated nucleic acid is SEQ ID NO: 54.

In another aspect, the present invention provides an isolated nucleic acid encoding a humanized IgG1 antibody as described herein, including embodiments. The humanized IgG1 antibody encoded by the isolated nucleic acid is described in detail throughout this specification (including the description above and the examples section). Thus, a humanized antibody encoded by an isolated nucleic acid includes all of the embodiments described herein. For example, the nucleic acid may encode at least one CDR, specific residues involved in epitope binding, or binding framework region residues. Thus, in some embodiments, the nucleic acid encodes a mouse L1 CDR as set forth in SEQ ID NO: 1, a mouse L2 CDR as set out in SEQ ID NO: 2, a mouse L3 CDR as set out in SEQ ID NO: 3. In some embodiments, the nucleic acid encodes a mouse H1 CDR as listed in SEQ ID NO: 4, a mouse H2 CDR as listed in SEQ ID NO: 5, and a mouse H3 CDR as listed in SEQ ID NO: 6. In some embodiments, In one embodiment, the nucleic acid encodes a mouse CDR L1 as shown in SEQ ID NO: 1, a mouse CDR L2 as listed in SEQ ID NO: 2, a mouse CDR L3 as listed in SEQ ID NO: 3, a mouse CDR H1 as listed in SEQ ID NO: 4, mouse H2 CDR as set forth in SEQ ID NO: 5, or mouse H3 CDR as set out in SEQ ID NO: 6. In some embodiments, the nucleic acid encodes a mouse L1 CDR as set out in SEQ ID NO. : 1, mouse CDR L2 as listed in SEQ ID NO: 2, mouse CDR L3 as listed in SEQ ID NO: 3, mouse CDR H1 as listed in SEQ ID NO: 4, mouse CDR H2 as listed in SEQ ID NO: 5, and mouse H3 CDRs as indicated in SEQ ID NO: 6.

In another aspect, the present invention provides an isolated nucleic acid encoding a humanized IgG4 antibody as described herein, including embodiments. The humanized IgG4 antibody encoded by the isolated nucleic acid is described in detail throughout this specification (including the description above and the examples section). Thus, a humanized antibody encoded by an isolated nucleic acid includes all of the embodiments described herein. For example, the nucleic acid may encode at least one CDR, specific residues involved in epitope binding, or binding framework region residues. Thus, in some embodiments, the nucleic acid encodes a murine CDR L1 as set forth in SEQ ID NO: 1, a murine CDR L2 as set forth in SEQ ID NO: 2, a murine CDR L3 as set forth in SEQ ID NO: 3. in some embodiments, the nucleic acid encodes a mouse H1 CDR as listed in SEQ ID NO: 4, a mouse H2 CDR as listed in SEQ ID NO: 5, and a mouse H3 CDR as listed in SEQ ID NO: 6. In some embodiments, nucleic acid encodes a mouse CDR L1 as listed in SEQ ID NO: 1, a mouse CDR L2 as listed in SEQ ID NO: 2, a mouse CDR L3 as listed in SEQ ID NO: 3, a mouse CDR H1 as listed in SEQ ID NO: 4, a mouse H2 CDR as set forth in SEQ ID NO: 5, or a mouse H3 CDR as set out in SEQ ID NO: 6. In some embodiments, the nucleic acid encodes a mouse L1 CDR as set out in SEQ ID NO: 1, mouse CDR L2 as listed in SEQ ID NO: 2, mouse CDR L3 as listed in SEQ ID NO: 3, mouse CDR H1 as listed in SEQ ID NO: 4, mouse CDR H2 as listed in SEQ ID NO: 5, and mouse H3 CDRs as indicated in SEQ ID NO: 6.

Pharmaceutical compositions

In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the humanized 1E9 antibody described herein, including embodiments thereof, and a pharmaceutically acceptable excipient.

In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a humanized IgG1 antibody as described herein, including embodiments thereof, and a pharmaceutically acceptable excipient.

In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a humanized IgG4 antibody as described herein, including embodiments thereof, and a pharmaceutically acceptable excipient.

A therapeutically effective amount, as described herein, refers to an amount effective to achieve its intended purpose. The actual amount effective for a particular application may depend, inter alia, on the condition being treated. When administered in methods of treating a disease, the pharmaceutical compositions described herein may contain an amount of an active humanized antibody effective to achieve a desired result, such as modulating the activity of a target molecule (eg, CD73), and/or reducing, eliminating, or delaying symptoms of progression. disease (e.g. cancer). Determining a therapeutically effective amount of a humanized antibody provided herein is entirely within the purview of those skilled in the art, especially in light of the detailed description herein.

Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate or acetate at pH typically 5.0-8.0, most commonly 6.0-7.0 ; salts such as sodium chloride, potassium chloride, etc. to obtain isotonicity; antioxidants, preservatives, low molecular weight polypeptides, proteins, hydro

- 43 042795 philic polymers such as polysorbate 80, amino acids such as glycine, carbohydrates, chelating agents, sugars, and other standard ingredients known to those skilled in the art (Remington's Pharmaceutical Science ^16th edition, Osol, A. Ed. 1980). mAbs are typically present at a concentration of 0.1-100 mg/ml, such as 1-10 mg/ml or 10-50 mg/ml, such as 5, 10, 20, 30, 40, 50, or 60 mg/ml ml.

A pharmaceutical composition containing a humanized antibody as described herein can be administered by a variety of methods known in the art. The route and/or route of administration will vary depending on the desired results. In some embodiments, the administration is intravenous, intramuscular, intraperitoneal, or subcutaneous, or administration in the vicinity of the target site. Pharmaceutically acceptable excipients may be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (eg, by injection or infusion).

Humanized antibody pharmaceutical compositions can be prepared according to methods well known and generally accepted in the art (See, for example, Remington: T The Science and Practice of Pharmacy, Mack Publishing Co., ^20th ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978). Pharmaceutical compositions are preferably manufactured under GMP conditions. Typically, a therapeutically effective dose or effective dose of a humanized antibody is used in the pharmaceutical compositions of the invention. The present humanized antibodies can be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art. Dosing regimens are adjusted to provide the optimal desired response (eg, therapeutic response). For example, a single bolus may be administered, multiple divided doses may be administered over time, or the dose may be proportionally increased or decreased as dictated by the requirements of the therapeutic situation. May provide advantages in making humanized antibody formulations in combination with other drugs or agents. May provide the advantage of formulating parenteral compositions in unit dosage forms for ease of administration and uniformity of dosing. Unit dosage form, as used herein, refers to physically discrete units suitable as unit doses for subjects to be treated; each unit contains a predetermined amount of humanized antibody calculated to produce the desired therapeutic effect, in combination with the required pharmaceutical excipient.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and route of administration, without being toxic to the patient. The selected dose level depends on a variety of pharmacokinetic factors, including the activity of the specific compositions used in the present invention, the route of administration, the time of administration, the rate of elimination of the particular antibody used, the duration of treatment, other drugs, compounds and/or materials used in combination with the specific used compositions, age, sex, weight, condition, general health and previous history of the patient being treated, and the like.

The physician or veterinarian may begin dosing the humanized antibodies of the invention used in a pharmaceutical composition at levels below those necessary to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. In general, effective dosages of the compositions of the present invention will vary depending on a variety of different factors, including the particular disease or condition being treated, routes of administration, target site, physiological state of the patient, whether the patient is human or animal, and other drugs administered. , and whether the treatment is prophylactic or therapeutic. Doses for treatment should be titrated to optimize safety and efficacy. For antibody administration, the dose is in the range of about 0.0001 to 100 mg/kg, and more typically 0.01 to 5 mg/kg of host body weight. For example, dosages may be 1 mg/kg body weight or 10 mg/kg body weight, or in the range of 1-10 mg/kg. An exemplary treatment regimen is administered once every two weeks, or once a month, or once every 36 months.

The humanized antibody provided herein can be administered in many situations. The intervals between single doses can be weekly, monthly or yearly. The intervals may also be uneven, as shown by measuring levels of the humanized antibody in the patient's blood. In some methods, the dose is adjusted to achieve a plasma antibody concentration of 1-1000 μg/ml, and in some methods 25-300 μg/ml. Alternatively, the antibody may be administered in the form of a sustained release formulation, in which case less frequent administration is necessary. The dose and frequency vary depending on the half-life of the antibody in the patient's body. Generally, humanized antibodies have been shown to have longer half-lives than chimeras.

- 44 042795 antibodies and non-human antibodies. The dose and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dose is administered at relatively infrequent intervals over a long period of time. Some patients continue to be treated for the rest of their lives. In therapeutic applications, a relatively high dose is sometimes needed at relatively short intervals until the progression of the disease is reduced or stopped, and preferably until the patient shows partial or complete relief of the symptoms of the disease. After that, the patient can be treated in a prophylactic regimen.

Ways

In one aspect, the present invention relates to a method of treating cancer in a subject in need thereof. The method includes administering to a subject a therapeutically effective amount of a humanized 1E9 antibody provided herein, including embodiments thereof, thereby treating the subject's cancer. In some embodiments, the cancer is a lymphoid cancer.

In one aspect, the present invention relates to a method of treating cancer in a subject in need thereof. The method includes administering to a subject a therapeutically effective amount of a humanized IgG1 antibody provided herein, including embodiments thereof, thereby treating a cancer in the subject. In some embodiments, the cancer is a lymphoid cancer.

Methods of inhibition

In one aspect, the present invention relates to a method for inhibiting cell proliferation. The method includes:

(i) contacting a cell with a humanized IgG1 antibody as described herein, including embodiments thereof, thereby obtaining a contacted cell;

(ii) the humanized IgG1 antibody is allowed to bind the CD73 antigen on the contacted cell, thereby inhibiting cell proliferation. In some embodiments, the cell is a lymphoid cell. In some embodiments, the lymphoid cell is a T cell.

In one aspect, the present invention relates to a method for inhibiting cell proliferation. The method includes:

(i) contacting a cell with a humanized IgG4 antibody as described herein, including embodiments thereof, thereby obtaining a contacted cell;

(ii) the humanized IgG4 antibody is allowed to bind the CD73 antigen on the contacted cell, thereby inhibiting cell proliferation.

In some embodiments, the cell is a lymphoid cell. In some embodiments, the lymphoid cell is a T cell.

Detection methods

In one aspect, the present invention provides a method for detecting a humanized 1E9 antibody bound to the CD73 antigen.

The method includes:

(i) contacting the humanized 1E9 antibody with the CD73 antigen at a pH less than about 7.5; and (ii) detecting the binding of the humanized 1E9 antibody to the CD73 antigen.

In some embodiments, the pH is from about 6.0 to about 7.0. In some embodiments, the pH is about 6.7. In some embodiments, the pH is about 6.3. In some embodiments, detection of binding from step (ii) includes detection of inhibition of CD73 catalytic activity. In some embodiments, the CD73 antigen forms part of the cell. In some embodiments, the CD73 antigen is associated with a solid support. In some embodiments, the humanized 1E9 antibody includes a detectable group.

Ways to activate T cells

The present invention relates to methods for activating an immunosuppressed (non-activated, non-proliferating) T cell in a cancerous environment. Thus, one aspect relates to a method for activating an immunosuppressed T cell.

The method includes:

(i) contacting a T cell with a humanized 1E9 antibody as described herein, including embodiments thereof, thereby obtaining a contacted T cell;

(ii) the humanized 1E9 antibody is allowed to bind the CD73 antigen on the contacted T cell, thereby activating the immunosuppressed T cell.

- 45 042795

In some embodiments, the implementation of the T cell is in the environment of a malignant tumor. In some embodiments, secretion of IFN-gamma by the contacted T cell is increased compared to the absence of the antibody. In some embodiments, the proliferation of the contacted T cell is increased compared to the absence of the antibody. An immunosuppressed T cell, as described herein, is a T cell that is in the environment of a cancer (in close proximity and/or in physiological contact with a cancer cell or solid tumor) that does not proliferate or secrete detectable amounts of cytokines or expresses cell surface markers characteristic of activated T cells (eg, IFN-gamma, CD25, CD38).

Methods of combined treatment

Methods of treatment presented in the present description, including variants of its implementation, may include the introduction of a second drug. Thus, treatments as described herein include administering a humanized 1E9 antibody as described herein or a humanized IgG1 or IgG4 antibody as described herein in combination with a second drug. The second drug may be any composition that can be used to treat or prevent cancer.

In one aspect, the present invention relates to a method of treating cancer in a subject in need thereof. The method includes administering to a subject a therapeutically effective amount of a humanized 1E9 antibody provided herein, including embodiments thereof, and an effective amount of a second drug, thereby treating the subject's cancer.

In another aspect, the present invention relates to a method of treating cancer in a subject in need thereof. The method includes administering to a subject a therapeutically effective amount of a humanized IgG1 antibody provided herein, including embodiments thereof, and an effective amount of a second drug, thereby treating the subject's cancer.

In another aspect, the present invention relates to a method of treating cancer in a subject in need thereof. The method includes administering to a subject a therapeutically effective amount of a humanized IgG4 antibody provided herein, including embodiments thereof, and an effective amount of a second drug, thereby treating the subject's cancer.

The second drug that can be used for the methods presented herein may be a compound, drug, antagonist, inhibitor, or modulator having antineoplastic properties or the ability to inhibit cell growth or proliferation. In some embodiments, the implementation of the second drug is a chemotherapeutic agent. A chemotherapeutic agent or chemotherapeutic agent is used according to its simple ordinary meaning, and refers to a chemical composition or compound having antineoplastic properties or the ability to inhibit cell growth or proliferation. In some embodiments, the implementation of the second drug is a radiotherapy. In some embodiments, the second drug is an FDA-approved or similar non-US regulatory agency for the treatment of cancer.

In some embodiments, the second drug is a compound. In some embodiments, the compound is a purine receptor antagonist. In some embodiments, the compound is an adenosine A _2A receptor antagonist or an adenosine A _2B receptor antagonist. In some embodiments, the compound is an A _2A adenosine receptor antagonist. In some embodiments, the compound is an adenosine A _2B receptor antagonist. In some embodiments, the compound is any of the compounds described in US Pat. In some embodiments, the compound is a thienopyrimidine compound. In some embodiments, the compound has the structure of the formula:

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C20H21N ₇ O ₃ Mol. mass: 407.43 (I).

The term adenosine A2A receptor as used herein includes any of the recombinant or natural forms of the adenosine A2A receptor (ADORA2A) or variants or homologues thereof that retain the activity of the ADORA2A protein (e.g., in the range of at least 50, 80, 90, 95, 96, 97, 98, 99 or 100% potency compared to ADORA2A). In some aspects, variants or homologues have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of a sequence (e.g., portions of 50, 100, 150, or 200 contiguous amino acids) at compared with the natural ADORA2A polypeptide. In some embodiments, ADORA2A is a protein as identified by sequence reference in NCBI GI:5921992, a homolog or functional fragment thereof.

The term adenosine A _2B receptor as used herein includes any of the recombinant or natural forms of the adenosine A _2B receptor (ADORA2B) or variants or homologues thereof that retain the activity of the ADORA2B protein (e.g., in the range of at least 50, 80, 90, 95, 96, 97, 98, 99 or 100% activity compared to ADORA2B). In some aspects, variants or homologues have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of a sequence (e.g., portions of 50, 100, 150, or 200 contiguous amino acids) at compared with the natural ADORA2B polypeptide. In some embodiments, ADORA2B is a protein as identified by sequence reference in NCBI GI:4501951, a homolog or functional fragment thereof.

In some embodiments, the drug is a second humanized antibody. In some embodiments, the second humanized antibody is an antibody capable of binding programmed cell death protein ligand 1 (PDL1). In some embodiments, the second humanized antibody is atezolizumab. In some embodiments, the second humanized antibody is an antibody capable of binding programmed cell death 1 (PD-1) protein. In some embodiments, the implementation of the second humanized antibody is an antibody capable of binding to CTLA-4.

The term atezolizumab or MPDL3280A refers to a fully humanized, engineered monoclonal antibody of the IgG1 isotype against programmed cell death protein ligand 1 (PD-L1). In the conventional sense, atezolizumab refers to CAS registration number 1380723-44-3.

The term PDL-1 as used herein includes any of recombinant or natural forms of programmed cell death protein ligand 1 (PD-L1) or variants or homologues thereof that retain PDL-1 protein activity (e.g., within at least 50 , 80, 90, 95, 96, 97, 98, 99, or 100% activity compared to PDL-1). In some aspects, variants or homologues have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of a sequence (e.g., portions of 50, 100, 150, or 200 contiguous amino acids) at compared with natural PDL-1 polypeptide. In some embodiments, PDL-1 is a protein as identified by sequence reference in NCBI GI:390979639, a homolog or functional fragment thereof.

The term PD-1, as used herein, includes any of the recombinant or natural forms of the programmed cell death 1 (PD-1) protein, or variants or homologues thereof, that retain the activity of the PD-1 protein (e.g., in the range of at least 50, 80, 90, 95, 96, 97, 98, 99 or 100% activity compared to PD-1). In some aspects, variants or homologues have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of a sequence (e.g., portions of 50, 100, 150, or 200 contiguous amino acids) at compared with natural PD-1 polypeptide. In some embodiments, PD-1 is a protein as identified by sequence reference in NCBI GI:167857792, a homolog or functional fragment thereof.

The term CTLA-4 or CTLA-4 protein as used herein includes any of recombinant or natural forms of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) or variants or homologs thereof that retain CTLA-4 protein activity (e.g. , in pre

- 47 042795 cases of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to CTLA-4). In some aspects, variants or homologues have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of a sequence (e.g., portions of 50, 100, 150, or 200 contiguous amino acids) at compared with natural CTLA-4 polypeptide. In some embodiments, CTLA-4 is a protein as identified by sequence reference in NCBI GI:83700231, a homolog or functional fragment thereof.

In the present methods of treatment, additional drugs suitable for the disease (eg, cancer) being treated can be used. Thus, in some embodiments, the provided methods of treatment further comprise administering a second drug to a subject. Suitable additional drugs include, but are not limited to, analgesics, anesthetics, analeptics, corticosteroids, anticholinergics, anticholinesterases, anticonvulsants, antineoplastic agents, allosteric inhibitors, anabolic steroids, antirheumatic agents, psychotherapeutic agents, neuroblocking agents, anti-inflammatory agents, anthelmintic agents, antibiotics, anticoagulants, antifungals, antihistamines, antimuscarinic agents, antimycobacterial agents, antiprotozoal agents, antiviral agents, dopaminergic agents, hematological agents, immunological agents, muscarinic agents, protease inhibitors, vitamins, growth factors and hormones. The choice of agent and dosage can be easily determined by one skilled in the art based on the disease being treated.

Combinations of agents or compositions can be administered either simultaneously (eg, in the form of a mixture), separately but simultaneously (eg, via separate intravenous lines), or sequentially (eg, one agent is administered first followed by a second agent). Thus, the term combination is used to refer to the joint, simultaneous or sequential administration of two or more agents or compositions. The course of treatment is best determined on an individual basis depending on the specific characteristics of the subject and the type of treatment chosen. Treatment, such as described herein, may be administered to the subject daily, twice daily, biweekly, monthly, or on any other acceptable basis that is therapeutically effective. Treatment can be carried out alone or in combination with any other treatment described in the present description or known in this field. The additional treatment may be given at the same time as the first treatment, at a different time, or on a completely different therapeutic schedule (eg, the first treatment may be given daily, while the additional treatment may be given weekly).

According to the methods provided herein, an effective amount of one or more of the drugs provided herein (i.e., a humanized 1E9 antibody or a humanized IgG1, or an IgG4 antibody, for example, in combination with a compound or a second humanized antibody). The terms effective amount and effective dose are used interchangeably. The term effective amount is defined as any amount necessary to obtain the desired physiological response (eg, reduction of inflammation).

Effective amounts and schedules for administering the agent can be empirically determined by one skilled in the art. Dosage ranges for administration are broad enough to provide the desired effect in which one or more symptoms of the disease or disorder are affected (eg, reduced or slowed down). The dose should not be so high as to cause significant adverse side effects such as unwanted cross-reactions, anaphylactic reactions, and the like. In general, the dosage may vary depending on age, condition, sex, type of disease, degree of disease or disorder, route of administration, or whether other drugs are included in the regimen, and may be determined by one of ordinary skill in the art. The dose can be adjusted by an individual therapist in case of any contraindications. Doses can be varied and can be administered in one or more doses daily, for one or more days. Guidance can be found in the literature for the appropriate dosing of these classes of pharmaceutical products. For example, for a given parameter, the effective amount may show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Efficiency can also be expressed as a multiple of increase or decrease. For example, a therapeutically effective amount may be at least 1.2-fold, 1.5-fold, 2-fold, 5-fold, or greater than a control. The exact dosage and formulation may depend on the goal of treatment and may be determined by those skilled in the art using known methods (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, ^20th Edition, Gennaro, Editor (2003), and Pickar, Dosage Calculations (1999)).

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Examples

Example 1

Switching the anti-CD73 antibody (1E9) to the human IgG1 isotype abolishes the direct effects on T cell activation seen with the mouse IgG3 isotype.

An antibody derived from a cell line derived from hybridoma 1E9 has the mIgG3 isotype. This antibody has been shown to synergize with PMA to activate T cell proliferation, IL-2 secretion, and upregulate IL-2 receptor expression (PMID: 2550543). This effect is thought to be through a direct signaling mechanism, since 1E9 mediates these effects on cells expressing catalytically inactive CD73 or transmembrane-attached CD73 to the cell membrane, in contrast to the GPI-anchoring mechanism used endogenously (PMID: 9113412, 7697732, 8027539). Applicants moved the variable regions from 1E9 into a human IgG1 scaffold, producing a chimeric 1E9 antibody. Chimeric 1E9 does not mediate activation of T cell proliferation or the IL-2 receptor (CD25 expression). Thus, Applicants have discovered that isotype switching can alter antibody-mediated effects on CD73 signaling.

Cellular assay to evaluate the catalytic activity of CD73

CD73 is expressed on the cell surface and is an ectonucleotidase that hydrolyzes AMP to adenosine and phosphate. To evaluate the catalytic activity of CD73 and the ability of Applicants' anti-CD73 antibodies to inhibit this activity, Applicants used a cellular assay. Cells endogenously expressing CD73 were incubated with anti-CD73 antibodies or isotype control antibody over a range of concentrations at 37° C. before adding AMP. Cells were incubated with AMP at 37°C for 20 min. Medium phosphate levels were measured using a commercially available reagent (Sensolyte MG phosphate assay kit, AnaSpec) and were directly proportional to CD73 activity. This assay was adapted from the literature and has previously been described for use in measuring CD73 activity (PMID: 21506751). To the knowledge of applicants, this assay has always been performed at a physiological pH of approximately 7.2. Surprisingly, Applicants were able to show that if the assay was performed at a lower pH to screen for anti-CD73 activity, the ability to block CD73 activity was retained at the lower pH. Strong activity at a slightly acidic pH may be a desirable property for a therapeutic antibody to be used for solid tumor indications, since the microenvironment of a solid tumor is known to be slightly acidic. Applicants have found that some antibodies (CPX-002, CPX-005, CPX-006) retain CD73 blocking activity at lower pH (6.3 or 6.7), while other antibodies (CPX-003, CPX -004) lose activity at lower pH.

Affinity measurements

Applicants obtained affinity measurements for 5 humanized CD73 candidate antibodies (CPX-003, CPX-004, CPX-005, CPX-006, CPX-007), as well as for a chimeric antibody (CPX-002), to measure the binding of these antibodies with CD73. The results of these affinity measurements are summarized in the table.

Table. Affinity measurements of humanized and chimeric anti-CD73 antibodies

Specific chain associated with highest affinity for CD73 and best activity at low pH

The two humanized antibodies (CPX-005, CPX-006) have higher affinity for CD73 and improved activity for inhibiting CD73 activity compared to other candidates. These two antibodies use the same heavy chain and differ only in the light chain. Thus, this particular heavy chain may be important to achieve high affinity and strong inhibition of CD73 activity. In some embodiments, the humanized heavy chain variable region comprises the sequence from SEQ ID NO: 7. In some embodiments, the humanized heavy chain variable region is the sequence from SEQ ID NO: 7.

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Example 2 Humanization of clone BAP094-01

Applicants have completed the construction of a library for the humanization of clone BAP094-01 (chimeric 1E9). Double-stranded DNA fragments encoding the BAPO94-O1 light chain and heavy chain CDR sequences (SEQ ID NO: 28 and SEQ ID NO: 29, respectively) were combined with human framework pools. The full length variable domains were then cloned into a mammalian expression vector. The light chain variable domains were cloned in-frame with the secretion signal and the human kappa constant domain. The heavy chain variable domains were cloned in-frame with the leader sequence and human IgG1 constant domain. Library quality (diversity of synthesized variable domains) was confirmed by sequencing (data not shown).

Screening of Humanized Variants

Received arrays of humanized clones in 96-well tablets. Each plate also contains two wells of a positive control (BAP094-01, ie chimeric 1E9) and a negative control (vector only). Plasmid DNA was prepared for each plate and transfected into CHO-S cells in a 96-well format. The supernatant was collected 48 hours after transfection. The IgG concentration was determined using a human IgG quantification ELISA method. Binding of humanized clones to CD73 expressed on the surface of MDA-MB-231 cells was determined using a cell-based ELISA. The clones with the best response from the primary screen were re-arrayed, re-transfected, and re-screened by cell-based ELISA (FIG. 5).

Sequence analysis of best response humanized variants

The light chain and heavy chain variable domains of the selected best responding humanized clone variants were sequenced and aligned with the original murine clone BAP094-01 (1E9) sequences. Sequence analysis indicated that there were four different heavy chains and 5 different light chains in the 10 clones with the best response (FIGS. 4A and 4B). Each clone with the best response has a unique combination of humanized light and heavy chain.

Formal sequence listing

SEQ ID NO:1: RASKNVSTSGYSYMH

SEQ ID NO:2: LASNLES

SEQ ID NO:3: QHSRELPFT

SEQ ID NO:4: GYTFTSYWIT

SEQ ID NO:5: PGSGNTNYNEKFKT

SEQ ID NO:6: EGGLTTEDYALDY

SEQ ID NO:7:

QVQLVQSGAEVEKPGASVKVSCKASGYTFTSYWITWVRQAPGQGLEWMGDIYPGSGTNTN

YNEKFKTRVTITADKSTSTAYMELSSLRSEDTAVYYCAKEGGLTTEDYALDYWGQGTLVTV

BAP094-hum01-LC SEQ ID NO: 8 :

GCCATCCAGTTGACCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCA

CTTGCAGGGCCAGCAAAAATGTCAGTACATCTGGCTATAGTTATATGCACTGGTATCAGCAGAA

ACCAGGGAAAGCTCCTAAGCTCCTGATCTATCTTGCATCCAACCTAGAATCTGGGATCCCACCT

CGGTTCAGTGGCAGCGGGTATGGAACAGATTTTACCCTCACAATTAATAACATAGAATCTGAGG

ATGCTGCATATTACTTCTGTCAGCACAGTAGGGAGCTTCCATTCACGTTCGGCCAAGGGACCAA

GGTGGAAAATCAAA

BAP094-hum02-LC SEQ ID NO:9:

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GAAATTGTGCTGACTCAGTCTCCAGACTTTCAGTCTGTGACTCCAAAGGAGAAAGTCACCATCA CCTGCAGGGCCAGCAAAAATGTCAGTACATCTGGCTATAGTTATATGCACTGGTATCAGCAGAA ACCAGGGAAAGCTCCTAAGCTCCTGATCTATCTTGCATCCAACCTAGAATCTGGGATCCCACCT CGATTCAGTGGCAGCGGGTATGGAACAGATTTTACCCTCACAATTAATAACATAGAATCTGAGG ATGCTGCATATTACTTCTGTCAGCACAGTAGGGAGCTTCCATTCACGTTCGGCCAAGGGACCAA GGTGGAAATCAAA

BAP094-humO3-LC, CPX-003 SEQ ID NO:10

GAAATTGTGCTGACTCAGTCTCCAGACTTTCAGTCTGTGACTCCAAAGGAGAAAGTCACCATCA CCTGCAGGGCCAGCAAAAATGTCAGTACATCTGGCTATAGTTATATGCACTGGTATCAGCAGAA ACCAGGGAAAGCTCCTAAGCTCCTGATCTATCTTGCATCCAACCTAGAATCTGGGATCCCACCT CGATTCAGTGGCAGCGGGTATGGAACAGATTTTACCCTCACAATTAATAACATAGAATCTGAGG ATGCTGCATATTACTTCTGTCAGCACAGTAGGGAGCTTCCATTCACGTTCGGCCAAGGGACCAA GGTGGAAATCAAA

BAP094-hum04-LC SEQ ID NO:11

GAAATTGTGCTGACTCAGTCTCCAGACTTTCAGTCTGTGACTCCAAAGGAGAAAGTCACCATCA CCTGCAGGGCCAGCAAAAATGTCAGTACATCTGGCTATAGTTATATGCACTGGTATCAGCAGAA ACCAGGGAAAGCTCCTAAGCTCCTGATCTATCTTGCATCCAACCTAGAATCTGGGATCCCACCT CGATTCAGTGGCAGCGGGTATGGAACAGATTTTACCCTCACAATTAATAACATAGAATCTGAGG ATGCTGCATATTACTTCTGTCAGCACAGTAGGGAGCTTCCATTCACGTTCGGCCAAGGGACCAA GGTGGAAATCAAA

BAP094-humO5-LC, CPX-004 SEQ ID NO:12

GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCT CCTGCAGGGCCAGCAAAAATGTCAGTACATCTGGCTATAGTTATATGCACTGGTACCAGCAGAA ACCTGGCCAGGCTCCCAGGCTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGATCCCACCT CGATTCAGTGGCAGCGGGTATGGAACAGATTTTACCCTCACAATTAATAACATAGAATCTGAGG ATGCTGCATATTACTTCTGTCAGCACAGTAGGGAGCTTCCATTCACGTTCGGCCAAGGGACCAA GGTGGAAATCAAA

BAP094-humO6-LC, CPX-005 SEQ ID NO:13

GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCA CTTGCAGGGCCAGCAAAAATGTCAGTACATCTGGCTATAGTTATATGCACTGGTACCAGCAGAA ACCTGGCCAGGCTCCCAGGCTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCCTCG AGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACCTTTACCATCAGTAGCCTGGAAGCTGAAG ATGCTGCAACATATTACTGTCAGCACAGTAGGGAGCTTCCATTCACGTTCGGCCAAGGGACCAA GGTGGAAATCAAA

BAP094-hum07-LC, CPX-006, CPX-007 SEQ ID NO:14

GAAATTGTGTTGACACAAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCT

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CCTGCAGGGCCACAAAAATGTCAGTACATCTGGCTATAGTTATATGCACTGGTACCAGCAGAA ACCTGGCCAGGCTCCCAGGCTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGATCCCACCT CGATTCAGTGGCAGCGGGTATGGAACAGATTTTACCCTCACAATTAATAACATAGAATCTGAGG

BAP094-humO8-LC SEQ ID NO:15:

GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCT CCTGCAGGGCCAGCAAAAATGTCAGTACATCTGGCTATAGTTATATGCACTGGTACCAGCAGAA ACCTGGCCAGGCTCCCAGGCTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGATCCCACCT CGATTCAGTGGCAGCGGGTATGGAACAGATTTTACCCTCACAATTAATAACATAGAATCTGAGG ATGCTGCATATTACTTCTGTCAGCACAGTAGGGAGCTTCCATTCACGTTCGGCCAAGGGACCAA GGTGGAAATCAAA

BAP094-humO9-LC SEQ ID NO :16:

GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCT CCTGCAGGGCCAGCAAAAATGTCAGTACATCTGGCTATAGTTATATGCACTGGTACCAGCAGAA ACCTGGCCAGGCTCCCAGGCTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGATCCCACCT CGATTCAGTGGCAGCGGGTATGGAACAGATTTTACCCTCACAATTAATAACATAGAATCTGAGG ATGCTGCATATTACTTCTGTCAGCACAGTAGGGAGCTTCCATTCACGTTCGGCCAAGGGACCAA GGTGGAAATCAAA

BAP094-humlO-LC SEQ ID NO:17:

GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCT CCTGCAGGGCCAGCAAAAATGTCAGTACATCTGGCTATAGTTATATGCACTGGTACCAGCAGAA ACCTGGCCAGGCTCCCAGGCTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGATCCCACCT CGATTCAGTGGCAGCGGGTATGGAACAGATTTTACCCTCACAATTAATAACATAGAATCTGAGG ATGCTGCATATTACTTCTGTCAGCACAGTAGGGAGCTTCCATTCACGTTCGGCCAAGGGACCAA GGTGGAAATCAAA

BAP094-humOl-HC SEQ ID NO:18:

CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGGAGAAGCCTGGGGCCTCAGTGAAGGTCTCCT GCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATAACCTGGGTGCGACAGGCCCCTGGACA AGGGCTTGAGTGGATGGGTGATATTTATCCTGGTAGTGGTAATACTAACTACAATGAGAAGTTC AAGACCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCC TGAGATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGAGGGAGGTCTTACTACGGAGGATTA TGCTTTGGACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCA

BAP094-humO2-HC SEQ ID NO:19:

CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGGAGAAGCCTGGGGCCTCAGTGAAGGTCTCCT GCAAGGCTTTCTGGCTACACCTTCACCAGCTACTGGATAACCTGGGTGCGACAGGCCCCTGGACA

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AGGGCTTGAGTGGATGGGTGATATTTATCCTGGTAGTGGTAATACTAACTACAATGAGAAGTTC

BAP094-humO3-HC, CPX-003, CPX-007 SEQ ID NO:20

CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCT GCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATAACCTGGGTGCGACAGGCTCGTGGACA ACGCCTTGAGTGGATAGGTGATATTTATCCTGGTAGTGGTAATACTAACTACAATGAGAAGTTC AAGACCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCC TGAGATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGAGGGAGGTCTTACTACGGAGGATTA TGCTTTGGACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCA

BAP094-hum04-HC SEQ ID NO:21

GAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCTACAGTGAAAATCTCCT GCAAGGTTTCTGGCTACACCTTCACCAGCTACTGGATAACCTGGATCCGCCAGCCCCCAGGGAA GGGGCTGGAGTGGATTGGTGATATTTATCCTGGTAGTGGTAATACTAACTACAATGAGAAGTTC AAGACCAGAGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCC TGAAGGCCTCGGACACCGCCATGTATTACTGTGCAAAAGAGGGAGGTCTTACTACGGAGGATTA TGCTTTGGACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCA

BAP094-hum05-HC, CPX-004 SEQ ID NO:22

GAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCTACAGTGAAAATCTCCT GCAAGGTTTCTGGCTACACCTTCACCAGCTACTGGATAACCTGGATCCGCCAGCCCCCAGGGAA GGGGCTGGAGTGGATTGGTGATATTTATCCTGGTAGTGGTAATACTAACTACAATGAGAAGTTC AAGACCAGAGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCC TGAAGGCCTCGGACACCGCCATGTATTACTGTGCAAAAGAGGGAGGTCTTACTACGGAGGATTA TGCTTTGGACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCA

BAP094-hum06-HC, CPX-005, CPX-006 SEQ ID NO:23

CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGGAGAAGCCTGGGGCCTCAGTGAAGGTCTCCT GCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATAACCTGGGTGCGACAGGCCCCTGGACA AGGGCTTGAGTGGATGGGTGATATTTATCCTGGTAGTGGTAATACTAACTACAATGAGAAGTTC AAGACCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCC TGAGATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGAGGGAGGTCTTACTACGGAGGATTA TGCTTTGGACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCA

BAP094-hum07-HC SEQ ID NO:24

CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGGAGAAGCCTGGGGCCTCAGTGAAGGTCTCCT GCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATAACCTGGGTGCGACAGGCCCCTGGACA AGGGCTTGAGTGGATGGGTGATATTTATCCTGGTAGTGGTAATACTAACTACAATGAGAAGTTC

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AAGACCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCC TGAGATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGAGGGAGGTCTTACTACGGAGGATTA TGCTTTGGACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCA

BAP094-humO8-HC SEQ ID NO:25:

GAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCTACAGTGAAAATCTCCT GCAAGGTTTCTGGCTACACCTTCACCAGCTACTGGATAACCTGGGTGCGACAGGCCCCTGGACA AGGGCTTGAGTGGATGGGTGATATTTATCCTGGTAGTGGTAATACTAACTACAATGAGAAGTTC AAGACCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCC TGAGATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGAGGGAGGTCTTACTACGGAGGATTA TGCTTTGGACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCA

BAP094-hum09-HC SEQ ID NO :26:

CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCT GCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATAACCTGGGTGCGACAGGCTCGTGGACA ACGCCTTGAGTGGATAGGTGATATTTATCCTGGTAGTGGTAATACTAACTACAATGAGAAGTTC AAGACCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCC TGAGATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGAGGGAGGTCTTACTACGGAGGATTA TGCTTTGGACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCA

BAP094-huml0-HC SEQ ID NO:27:

GAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCTACAGTGAAAATCTCCT GCAAGGTTTCTGGCTACACCTTCACCAGCTACTGGATAACCTGGATCCGCCAGCCCCCAGGGAA GGGGCTGGAGTGGATTGGTGATATTTATCCTGGTAGTGGTAATACTAACTACAATGAGAAGTTC AAGACCAGAGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCC TGAAGGCCTCGGACACCGCCATGTATTACTGTGCAAAAGAGGGAGGTCTTACTACGGAGGATTA TGCTTTGGACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCA

BAP094-01-LC CPX-002 (chimeric) SEQ ID NO:28:

GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCT CATGCAGGGCCAGCAAAAATGTCAGTACATCTGGCTATAGTTATATGCACTGGTACCAACAGAA ACCAGGACAGCCACCCAAACTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTACC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGG ATGCTGCAACCTATTACTGTCAGCACAGTAGGGAGCTTCCATTCACGTTCGGCTCGGGGACAAA GTTGGAAATAAAA

BAP094-01-HC CPX-002 (chimeric) SEQ ID NO:29:

CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTTGTGAAGCCTGGGGCTTCAGTGAAGATGTCCT GCAAGGCTTTCTGGCTACACCTTCACCAGCTACTGGATAACCTGGGTGAAGCAGAGGCCTGGACA AGGCCTTGAGTGGATTGGAGATATTTATCCTGGTAGTGGTAATACTAACTACAATGAGAAGTTC AAGACCAAGGCCACAGGCCAAGACTGTAGACACCTACATCCTCCA

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TGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAAAGAGGGAGGTCTTACTACGGAGGATTA TGCTTTGGACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCA

BAP094-01-LC SEQ IDNO:30

DIVLTQSPASLAVSLGQRATISCRASKNVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPT RFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGSGTKLEIK

BAP094-hum01-LC SEQ IDNO:31

AIQLTQSPSSLSASVGDRVTITCRASKNVSTSGYSYMHWYQQKPGKAPKLLIYLASNLESGIPP RFSGSGYGTDFTLTINNIESEDAAYYFCQHSRELPFTFGQGTKVEIK

BAP094-hum02-LC SEQ IDNO:32

EIVLTQSPDFQSVTPKEKVTITCRASKNVSTSGYSYMHWYQQKPGKAPKLLIYLASNLESGIPP RFSGSGYGTDFTLTINNIESEDAAYYFCQHSRELPFTFGQGTKVEIK

BAP094-humO3-LC SEQ IDNO:33

EIVLTQSPDFQSVTPKEKVTITCRASKNVSTSGYSYMHWYQQKPGKAPKLLIYLASNLESGIPP RFSGSGYGTDFTLTINNIESEDAAYYFCQHSRELPFTFGQGTKVEI

BAP094-hum04-LC SEQ IDNO:34

EIVLTQSPDFQSVTPKEKVTITCRASKNVSTSGYSYMHWYQQKPGKAPKLLIYLASNLESGIPP RFSGSGYGTDFTLTINNIESEDAAYYFCQHSRELPFTFGQGTKVEIK

BAP094-humO5-LC SEQ IDNO:35

DWMTQSPLSLPVTLGQPASISCRASKNVSTSGYSYMHWYQQKPGQAPRLLIYLASNLESGIPP RFSGSGYGTDFTLTINNIESEDAAYYFCQHSRELPFTFGQGTKVEIK

BAP094-humO6-LC SEQ IDNO:36

AIQLTQSPSSLSASVGDRVTITCRASKNVSTSGYSYMHWYQQKPGQAPRLLIYLASNLESGVPS RFSGSGSGTDFTFTISSLEAEDAATYYCQHSRELPFTFGQGTKVEIK

BAP094-hum07-LC SEQ IDNO:37

EIVLTQSPATLSLSPGERATLSCRASKNVSTSGYSYMHWYQQKPGQAPRLLIYLASNLESGIPP RFSGSGYGTDFTLTINNIESEDAAYYFCQHSRELPFTFGQGTKVEIK

BAP094-humO8-LC SEQ IDNO:38

EIVLTQSPATLSLSPGERATLSCRASKNVSTSGYSYMHWYQQKPGQAPRLLIYLASNLESGIPP RFSGSGYGTDFTLTINNIESEDAAYYFCQHSRELPFTFGQGTKVEIK

BAP094-humO9-LC SEQ IDNO:39

EIVLTQSPATLSLSPGERATLSCRASKNVSTSGYSYMHWYQQKPGQAPRLLIYLASNLESGIPP RFSGSGYGTDFTLTINNIESEDAAYYFCQHSRELPFTFGQGTKVEIK

BAP094-humlO-LC SEQ IDNO:40

EIVLTQSPATLSLSPGERATLSCRASKNVSTSGYSYMHWYQQKPGQAPRLLIYLASNLESGIPP RFSGSGYGTDFTLTINNIESEDAAYYFCQHSRELPFTFGQGTKVEIK

BAP094-01-HC SEQ IDNO:41

- 55 042795

QVQLQQPGAELVKPGASVKMSCKASGYTFTSYWITWVKQRPGQGLEWIGDIYPGSGNTNYNEKF KTKATLTVDTSSSTAYMQLSSLTSEDSAVYYCAKEGGLTTEDYALDYWGQGTLVTVSS

BAP094-hum01-HC SEQ ID NO:42:

QVQLVQSGAEVEKPGASVKVSCKASGYTFTSYWITWVRQAPGQGLEWMGDIYPGSGNTNYNEKF

KTRVTITADKSTSTAYMELSSLRSEDTAVYYCAKEGGLTTEDYALDYWGQGTLVTVSS

BAP094-hum02-HC SEQ ID NO:43:

QVQLVQSGAEVEKPGASVKVSCKASGYTFTSYWITWVRQAPGQGLEWMGDIYPGSGNTNYNEKF

KTRVTITADKSTSTAYMELSSLRSEDTAVYYCAKEGGLTTEDYALDYWGQGTLVTVSS

BAP094-hum03-HC SEQ ID NO:44:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWITWVRQARGQRLEWIGDIYPGSGNTNYNEKF

KTRVTITADKSTSTAYMELSSLRSEDTAVYYCAKEGGLTTEDYALDYWGQGTLVTVSS

BAP094-hum04-HC SEQ ID NO:45:

EVQLVQSGAEVKKPGATVKISCKVSGYTFTSYWITWIRQPPGKGLEWIGDIYPGSGNTNYNEKF

KTRVTISADKSISTAYLQWSSLKASDTAMYYCAKEGGLTTEDYALDYWGQGTLVTVSS

BAP094-hum05-HC SEQ ID NO :46:

EVQLVQSGAEVKKPGATVKISCKVSGYTFTSYWITWIRQPPGKGLEWIGDIYPGSGNTNYNEKF

KTRVTISADKSISTAYLQWSSLKASDTAMYYCAKEGGLTTEDYALDYWGQGTLVTVSS

BAP094-hum06-HC SEQ ID NO:47:

QVQLVQSGAEVEKPGASVKVSCKASGYTFTSYWITWVRQAPGQGLEWMGDIYPGSGNTNYNEKF

KTRVTITADKSTSTAYMELSSLRSEDTAVYYCAKEGGLTTEDYALDYWGQGTLVTVSS

BAP094-hum07-HC SEQ ID NO:48:

QVQLVQSGAEVEKPGASVKVSCKASGYTFTSYWITWVRQAPGQGLEWMGDIYPGSGNTNYNEKF

KTRVTITADKSTSTAYMELSSLRSEDTAVYYCAKEGGLTTEDYALDYWGQGTLVTVSS

BAP094-hum08-HC SEQ ID NO:49:

EVQLVQSGAEVKKPGATVKISCKVSGYTFTSYWITWVRQAPGQGLEWMGDIYPGSGNTNYNEKF

KTRVTITADKSTSTAYMELSSLRSEDTAVYYCAKEGGLTTEDYALDYWGQGTLVTVSS

BAP094-humO9-HC SEQ ID NO:50:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWITWVRQARGQRLEWIGDIYPGSGNTNYNEKF

KTRVTITADKSTSTAYMELSSLRSEDTAVYYCAKEGGLTTEDYALDYWGQGTLVTVSS

BAP094-huml0-HC SEQ ID NO:51:

EVQLVQSGAEVKKPGATVKISCKVSGYTFTSYWITWIRQPPGKGLEWIGDIYPGSGNTNYNEKF

KTRVTISADKSISTAYLQWSSLKASDTAMYYCAKEGGLTTEDYALDYWGQGTLVTVSS

CPX-006_HC (codon optimized)

SEQ ID NO:52:

AAGCTTGCCGCCACCATGGAATGGTCCTGGGTGTTCCTGTTCTTCCTGTCCGTGACCAC CGGCGTGCACTCCCAGGTGCAGCTGGTGCAGTCTGGCGCCGAGGTGGAAAAGCCTGGCGCCTCT

- 56 042795

GTGAAGGTGTCCTGCAAGGCCTCCGGCTACACCTTTACCAGCTACTGGATCACCTGGGTGCGAC AGGCTCCTGGACAGGGCCTGGAATGGATGGGCGACATCTACCCTGGCTCCGGCAACACCAACTA СAACGAGAAGT T CAAGACСC GCGT GAG CAT САСC GСCGAGAAGT СС AC С T ССАСC GСC TAGAT G GAACTGTCCTCCCTGCGGAGCGAGGACACCGCCGTGTACTACTGTGCTAAAGAGGGCGGCCTGA CCACCGAGGACTACGCCCTGGATTATTGGGGCCAGGGCACCCTCGTGACCGTGTCCTCTGCTTC TACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCTGGCGGCACAGCC GCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACTCTGGCG CCCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTC CTCCGTCGTGACTGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCAC AAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCT GTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCCTCTGTGTTTCTGTTCCCCCCAAAGCC CAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCAC GAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCA AGCCTAGAGAGGAACAGTACCAGTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCA GGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCTCTGCCTGCCCCCATC GAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTA GCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTC CGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCT GTGCTGGACTCCGACGGCTCATTCTTTCTGTACTCCAAGCTGACAGTGGACAAGTCCCGGTGGC AGCAGGGCAACGTGTTCTCCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAA GTCCCTGTCCCTGAGCCCCGGCAAGTGATGAATTC

CPX-006_HC (codon optimized) SEQ ID NO:53:

MEWSWVFLFFLSVTTGVHS

QVQLVQSGAEVEKPGASVKVSCKASGYTFTSYWITWVRQAPGQGLEWMGDIYPGSGNTN YNEKFKTRVTITADKSTSTAYMELSSLRSEDTAVYYCAKEGGLTTEDYALDYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRWSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK

CPX-006_LC (codon optimized) SEQ ID NO:54:

AAGCTTGCCGCCACCATGTCCGTGCCTACCCAGGTGCTGGGACTGCTGCTGCTGTGGCT

GACCGATGCCAGATGCGAGATCGTGCTGACCGATCCCCTGCCACCCTGTCACTGTCTCCAGGC GAGAGAGCCACCCTGAGCTGCCGGGCCTCCAAGAACGTGTCCACCTCCGGCTACTCCTACATGCACTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCTACCTGGCCTCCAACCTGGA

ATCCGGCATCCCCCCTAGATTCTCCGGCTCTGGCTACGGCACCGACTTCACCCTGACCATCAAC AACATCGAGTCCGAGGACGCCGCCTACTACTTCTGCCAGCACTCCAGAGAGCTGCCCTTCACCT TTGGCCAGGGCACCAAGGTGGAAATCAAGCGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCCC ACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTAC CCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAAT CCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCTACCCTGACCCTGTCCAA GGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCC GTGACCAAGTCTTTCAACCGGGGCGAGTGCTGATGAATTC

CPX-006_LC (codon optimized)

SEQ ID NO:55:

MSVPTQVLGLLLLWLTDARC

EIVLTQSPATLSPGERATLSCRASKNVSTSGYSYMHWYQQKPGQAPRLLIYLASNLE SGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQHSRELPFTFGQGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLECPVTHNSFQ

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Claims (18)

CLAIM 1. A humanized aHTu-CD73 antibody comprising a humanized light chain variable region and a humanized heavy chain variable region, wherein said humanized light chain variable region comprises SEQ ID NO: 36 or SEQ ID NO: 37, and wherein said humanized heavy chain variable region comprises SEQ ID NO: 7. 2. The humanized anti-CD37 antibody of claim 1, wherein said antibody is IgG4. 3. The humanized anti-CD73 antibody of claim 1, wherein said antibody is capable of binding the CD73 antigen with an equilibrium dissociation constant (K _D ) of about 0.5 to about 25 nM. 4. The humanized anti-CD73 antibody of claim 1, wherein said antibody is capable of binding the CD73 antigen at a pH of about 6.0 to about 7.0. 5. The humanized anti-CD73 antibody of claim 1 linked to the CD73 antigen. 6. The humanized anti-CD73 antibody of claim 5, wherein said CD73 antigen forms part of a cell. 7. The humanized anti-CD73 antibody of claim 6, wherein said cell is a cancerous cell. 8. A pharmaceutical composition containing a therapeutically effective amount of a humanized anti-CD73 antibody according to claim 1 and a pharmaceutically acceptable excipient. 9. A method of treating cancer in a patient, wherein said method comprises administering to said patient a therapeutically effective amount of the humanized anti-CD73 antibody of claim 1, thereby treating said patient's cancer. 10. A humanized anti-CD37 IgG1 antibody comprising a humanized light chain variable region and a humanized heavy chain variable region, wherein said humanized light chain variable region comprises a mouse L1 CDR with the sequence of SEQ ID NO: 1, a mouse L2 CDR with the sequence of SEQ ID NO: 2, a mouse L3 CDR of SEQ ID NO: 3, and wherein said humanized heavy chain variable region comprises a mouse H1 CDR of SEQ ID NO: 4, a mouse H2 CDR of SEQ ID NO: 5, and a mouse H3 CDR of SEQ ID NO: NO:6. 11. The humanized anti-CD37 IgG1 antibody of claim 10, wherein said humanized light chain variable region further comprises valine at position 2 according to Kabat, methionine at position corresponding to position 4 according to Kabat, leucine at position corresponding to position 9 according to Kabat, proline at Kabat position 12 and proline at Kabat position 18. 12. The humanized anti-CD37 IgG1 antibody of claim 10, wherein said humanized heavy chain variable region further comprises isoleucine at position corresponding to position 37 according to Kabat, proline at position corresponding to position 40 according to Kabat, lysine at position corresponding to position 43 according to Kabat. Kabat, serine at Kabat position 70, isoleucine at Kabat position 75, tryptophan at Kabat position 82, lysine at Kabat position 83, alanine at Kabat position 84 Kabat, serine at Kabat position 85 and methionine at Kabat position 89. 13. The humanized anti-CD37 IgG1 antibody of claim 10 linked to the CD73 antigen. 14. The humanized anti-CD37 IgG1 antibody of claim 13, wherein said CD73 antigen forms part of a cell. 15. The humanized anti-CD37 IgG1 antibody of claim 14, wherein said cell is a cancerous cell. 16. An isolated nucleic acid encoding a humanized anti-CD37 IgG1 antibody according to claim 10. 17. A pharmaceutical composition comprising a therapeutically effective amount of a humanized anti-CD37 IgG1 antibody according to claim 10 and a pharmaceutically acceptable excipient. 18. A method of treating cancer in a patient, wherein said method comprises administering to said patient a therapeutically effective amount of the humanized anti-CD37 IgG1 antibody of claim 10, thereby treating said patient's cancer. -