MELANIN ANTIBODIES AND USES THEREOF

CROSS-REFERENCE

[0001] This application claims the priority benefit of U.S. Provisional Patent Application Serial No. 62/558,230, filed on September 13, 2017, which is incorporated by reference in its entirety.

BACKGROUND

[0002] Melanoma, the most serious type of skin cancer, develops in the melanin-producing melanocytes. Melanoma can also originate in the uveal tract of the eye, in the mucosal epithelium lining the upper aero- digestive tract, and the intestinal tract. The American Cancer Society estimates that in 2017, about 87,000 new melanomas will be diagnosed and about 9,750 people are expected to die of melanoma, in the United States (https : //www, cancer, or g/cancer/melanoma- skin- cancer/about/key-statistics . html) . Globally, in 2012, melanoma occurred in about 232,000 people and resulted in about 55,000 deaths.

[0003] While stage 1 and 2 melanoma can be surgically treated, the aggressive metastatic nature of this malignancy provides a poor prognosis with estimated survival rates of 19%, 13%, and 9% at 3, 5, and 10 years, respectively, for patients with stage IV melanoma. (CM Balch, JE Gershenwald, SJ Soong , et al: Final version of 2009 AJCC melanoma staging and classification J Clin Oncol 27: 6199- 6206,2009). Approval by FDA of vemurafenib, which inhibits mutated B-RAF protein, offers hope for 40-60% melanoma patients carrying this mutation. Efforts to restore latent antitumor immunity have focused on monoclonal antibody (mAb)-based interventions targeting CTL antigen 4 (CTLA-4) (Hodi FS, O'Day SJ, McDermott DF, et al: Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 363:711-723, 2010) and programmed cell death protein 1 (PD-1) on T lymphocytes and its principal ligand (PD-L1) on tumor cells (Phillips GK, Atkins M. Therapeutic uses of anti-PD-1 and anti-PD-Ll antibodies. Int Immunol.

2015;27(l):39-46). With only a minority of patients experiencing long term progression free survival in response to either anti CTLA-4, or anti PD-1 pathway checkpoint inhibitor

immunotherapy, the significant risk of serious autoimmune toxicity associated with these agents, and the high costs of immunotherapy (Fellner, Chris. Ipilimumab (Yervoy) Prolongs Survival in Advanced Melanoma: Serious Side Effects and a Hefty Price Tag May Limit Its Use. Pharmacy &Therapeutics 2012:27(9):503-511), there remains an urgent need for other approaches to combat melanoma, especially metastatic melanoma.

SUMMARY

[0004] Provided herein are monoclonal antibodies that specifically bind to melanin. The antibodies may be chimeric or humanized. Also provided herein are methods of use and methods of making the antibodies described. For example, the melanin antibodies may be used therapeutically to treat or prevent melanoma.

[0005] Accordingly, in one aspect provided herein is a monoclonal antibody that specifically binds to melanin, wherein the antibody is chimeric or humanized.

[0006] In some embodiments, the antibody is chimeric. In some embodiments, the antibody is a chimeric mouse-human antibody. In some embodiments, the chimeric antibody comprises mouse variable regions and human constant regions. In some embodiments, the melanin antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1. In some

embodiments, the melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the melanin antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2.

[0007] In some embodiments, the antibody is humanized. In some embodiments, the antibody is a humanized form from the sequence of a mouse monoclonal antibody. In some embodiments, the antibody is a humanized form from a mouse 8C3 antibody. In some embodiments, the humanized melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the humanized melanin antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. In some embodiments, the humanized melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the humanized melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the humanized melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the humanized melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the humanized melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the humanized melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the heavy chain of the humanized melanin antibody comprises at least one of the CDR sequences of SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In some embodiments, the light chain of the humanized melanin antibody comprises at least one of the CDR sequences of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. In some embodiments, the heavy chain of the humanized melanin antibody comprises at least one of the CDR sequences of SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, and the light chain of the humanized melanin antibody comprises at least one of the CDR sequences of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. In some embodiments, the heavy chain of the humanized melanin antibody comprises the CDR sequences from SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, and/or the light chain comprises the CDR sequences from SEQ ID NO: 3 or SEQ ID NO: 4.

[0008] In some embodiments, the chimeric or humanized monoclonal melanin antibody is an antigen binding fragment.

[0009] In some embodiments, the chimeric or humanized monoclonal melanin antibody is a bispecific antibody. In some embodiments, the bispecific antibody comprises a first arm that targets melanin and a second arm that targets an antigen comprising an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is CTLA4, PD-1, or PD-L1.

[0010] In some embodiments, the chimeric or humanized monoclonal melanin antibody is conjugated to an agent. In some embodiments, the agent is a radionuclide. In some embodiments, the radionuclide is 213-Bi. In some embodiments, the radionuclide is 177-Lu. In some

embodiments, the agent is conjugated to the antibody through a linker. [0011] In a related aspect, provided herein is a pharmaceutical composition comprising any one of the chimeric or humanized monoclonal melanin antibodies provided herein, and a

pharmacologically acceptable carrier.

[0012] In another aspect, provided herein is a method for treating melanoma in a subject, comprising administering a therapeutically effective amount of any one of the monoclonal chimeric or humanized melanin antibodies or compositions comprising such antibodies, as described herein. In a related aspect, provided herein is a therapeutically effective amount of any one of the monoclonal chimeric or humanized melanin antibodies or compositions comprising such antibodies, as described herein for use in treating melanoma.

[0013] In some embodiments, the melanoma is metastasized. In some embodiments, the administration selectively induces the cell death of melanoma cells. In some embodiments, the method comprises administering to the subject an effective amount of at least one additional agent. In some embodiments, the agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is selected from CTLA-4, PD-1 , and PDL-1. In some embodiments, the antibody or composition is administered intravenously.

[0014] In another aspect, provided herein is a method of making a conjugated melanin antibody comprising conjugating any one of the monoclonal chimeric or humanized melanin antibodies described herein to an agent. In some embodiments, the agent is a radionuclide. In some embodiments, the radionuclide is 213-Bi. In some embodiments, the radionuclide is 177-Lu.

[0015] In another aspect provided herein are polynucleotides encoding the amino acid sequence of any one of the chimeric or humanized monoclonal melanin antibodies provided herein. In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 17. In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 18. In some embodiments, the polynucleotide has been codon optimized for expression in a human. Also provided herein are vectors comprising polynucleotides encoding the amino acid sequence of any one of the chimeric or humanized monoclonal melanin antibodies provided herein, and cell lines comprising such vectors. Also provided herein are clonal cell lines expressing any one of the chimeric or humanized monoclonal melanin antibodies provided herein [0016] In another aspect, provided herein is a kit comprising any one of the chimeric or humanized monoclonal antibodies or pharmaceutical compositions comprising such antibodies.

[0017] All of the above features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

[0018] For a better understanding of the invention, and to show how embodiments of the same may be carries into effect, reference will now be made, by way of example, to the accompanying diagrammatic drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIGS. 1 and 2 show the results of the binding of the chimeric 8C3 and humanized 8C3 antibodies to melanin, as assayed in vitro, in separate experiments.

[0020] FIG. 3 compares the binding of mouse 8C3 and mouse IgGl negative control antibodies to melanin from Sepia officinalis.

[0021] FIG. 4 provides schematic diagrams of the plasmids used for expression of the chimeric and humanized antibodies: FIG. 4A) pABl 1 8C3hIgGl 625.69.1, FIG. 4B) pAB2-8C3 hKappa- 625.48.2, FIG. 4C) AB2-8C3-HE-VK4-hKappa 625.85.1, FIG. 4D) pAB2-8C3-HE-VKlA- hKappa-625.85.2, FIG. 4E) pAB2-8C3-HE-VKlB-hKappa-625.85.3, FIG. 4F) pABl l-8C3-HE- VH3A-hIgGl 625.85.4, and FIG. 4G) pABl l-8C3-HE-VH3B-hIgGl 625.85.5.

[0022] FIG. 5 show alignments of the heavy chains of the antibodies described herein.

[0023] FIG. 6 show alignments of the light chains of the antibodies described herein.

[0024] FIG. 7 shows a representative C57BL/6 mouse bearing a B16-F10 melanoma tumor (indicated by the black circle) prior to undergoing any mAB-based anti-melanin or control treatment.

[0025] FIGS. 8A-8D depict the results of a biodistribution experiment that compared the uptake of radiolabeled melanin-binding antibodies in various organs to that of a non-specific human IgG antibody control at two different time points post-antibody injection (4 hours and 24 hours).

[0026] FIG. 9 shows the results of a tumor-to-blood ratio calculation, which provides a proxy measurement of the amount of radiolabeled melanin-binding antibodies that have bound the tumor. [0027] FIG. 10 is a graph depicting the biodistribution of 11 Hn-h8C3 HE-5 antibody in mice at pre-determined time points of 1, 2, 24, 48 and 72 hrs post- injection of the radiolabeled antibody.

[0028] FIGS. 11 A and 11B are graphs depicting tumor volume in mice treated with either: high dose of 213Bi-h8C3 HE-5, or low dose of 213Bi-h8C3 HE-5, or high dose of 177Lu-h8C3 HE-5, or low dose of 177Lu-h8C3 HE-5, or 80 μg unlabeled ("cold") h8C3 HE-5, or left untreated. Their tumors were measured every three days with electronic calipers to calculate the tumor volume.

[0029] FIG. 12 and FIG. 13 are a series of graphs depicting blood counts of 12A and 13A) white blood cells, 12B and 13B) red blood cells, 12C and 13C) and platelets in mice treated with either: high dose of 213Bi-h8C3 HE-5, or low dose of 213Bi-h8C3 HE-5, or high dose of 177Lu-h8C3 HE- 5, or low dose of 177Lu-h8C3 HE-5, or 80 μg unlabeled ("cold") h8C3 HE-5, or left untreated.

[0030] FIGS. 14A and 14B are a series of graphs depicting body weight of mice treated with either: high dose of 213Bi-h8C3 HE-5, or low dose of 213Bi-h8C3 HE-5, or high dose of 177Lu- h8C3 HE-5, or low dose of 177Lu-h8C3 HE-5, or 80 μg unlabeled ("cold") h8C3 HE-5, or left untreated.

[0031] FIG. 15 is a series of graphs depicting concentrations of blood analytes: 15A) alanine transaminase (ALT), 15B) aspartate transaminase (AST), 15C) urea, and 15D) creatinine, in mice treated with either: high dose of 213Bi-h8C3 HE-5, or low dose of 213Bi-h8C3 HE-5, or left untreated.

[0032] FIGS. 16A-16C are a series of graphs depicting changes in tumor volume in tumor- bearing mice randomized into groups of 8 and treated with either: single dose 400 μθ 213-h8C3 HE-5 on Day 0, or 400 μθί 213-h8C3 HE-5 on Day 0 and on Day 3, or 400 μθΐ 213-h8C3 HE-5 on Day 0, Day 3 and Day 7. On Day 16 mice in the single dose group were treated with another 400 μα 213-1ι803 ΗΕ-5 dose.

[0033] FIG. 17 is a graph depicting changes in body weight in tumor-bearing mice randomized into groups of 8 and treated with either: single dose 400 μθ 213-h8C3 HE-5 on Day 0, or 400 μθ 213-h8C3 HE-5 on Day 0 and on Day 3, or 400 μθΐ 213-h8C3 HE-5 on Day 0, Day 3 and Day 7. On Day 16 mice in the single dose group were treated with another 400 μθ 213-h8C3 HE-5 dose.

[0034] FIG. 18 is a series of graphs depicting blood counts of 18A white blood cells, 18B) red blood cells, 18C) and platelets in tumor-bearing mice randomized into groups of 8 and treated with either: single dose 400 μθΐ 213-h8C3 HE-5 on Day 0, or 400 μθΐ 213-h8C3 HE-5 on Day 0 and on Day 3, or 400 μθί 213-h8C3 HE-5 on Day 0, Day 3 and Day 7. On Day 16 mice in the single dose group were treated with another 400 μθ 213-h8C3 HE-5 dose.

[0035] FIG. 19 is a series of graphs depicting concentrations of blood analytes: 19A) alanine transaminase (ALT), 19B) aspartate transaminase (AST), 19C) urea, and 19D) creatinine, in tumor- bearing mice randomized into groups of 8 and treated with either: single dose 400 μθ 213-h8C3 HE-5 on Day 0, or 400 μθΐ 213-h8C3 HE-5 on Day 0 and on Day 3, or 400 μθΐ 213-h8C3 HE-5 on Day 0, Day 3 and Day 7. On Day 16 mice in the single dose group were treated with another 400 μα 213-1ι803 ΗΕ-5 dose.

[0036] FIG. 20 is a series of microSPECT/CT images of a mouse lh, 4h, 24h, 48h, 72h, 96h, and 216h post injection with 200 μθί 11 lln at a 5: 1 mCi/mg specific activity with a CHXA" conjugated h8C3 HE-5 antibody.

[0037] FIG. 21 is a graph depicting bulk pool cell growth.

[0038] FIG. 22 is a graph depicting the bulk pool titer profile as measured by ForteBio Octet Red.

[0039] FIG. 23 is a graph depicting the titer profile across 96-well plates of cells expressing antibody.

[0040] FIG. 24 is a graph depicting the titer profile of the 120-top expressing pools from FIG. 23 selected to grow in 24-well plates. Three super-pools were selected. Super-pool 1 was composed of the three highest expresser mini-pools with titers ranging from 106 to 129 μg/mL, the Super-pool 2 was composed of five mini-pools with titers ranging from 60 to 75 μg/mL and the Super-pool 3 was composed of seven mini-pools with titers ranging from 40 to 58 μg/mL.

[0041] FIG. 25 is a chart ranking the highest expressing pools from the 24-well plate screening. Three super-pools were selected. Super-pool 1 was composed of the three highest expresser mini- pools with titers ranging from 106 to 129 μg/mL, the Super-pool 2 was composed of five mini-pools with titers ranging from 60 to 75 μg/mL and the Super-pool 3 was composed of seven mini-pools with titers ranging from 40 to 58 μg/mL.

[0042] FIG. 26 is a graph depicting the growth curve of each super-pool.

[0043] FIG. 27 is a graph depicting the viability of each super-pool.

[0044] FIG. 28 is a graph depicting the titer profile of each super-pool. [0045] FIG. 29 is a graph depicting the titer profile of clones from the 24-well stage that were ranked based on expression levels measured on day 11 using a ForteBio Octet Red with a Protein A sensor and compared to a standard curve obtained with the 8C3 HE-5 antibody purified from the bulk pool.

[0046] FIG. 30 is a chart highlighting the 36 clones with the highest expression levels from the 24-well stage.

[0047] FIG. 31 is a chart highlighting the highest expressing clones: Clones 2-3H2, 2-3H11, 2- 11H12 and 2-20C3 with respective expression levels of 1.29 g/L, 1.27 g/L, 1.26 g/L, and 1.25 g/L.

DETAILED DESCRIPTION OF THE INVENTION

[0048] Provided herein are antibodies that specifically bind to melanin. The antibodies may be chimeric or humanized. Also provided herein are methods of use and methods of making the antibodies described. For example, the melanin antibodies may be used therapeutically to treat or prevent melanoma, comprising administering to a subject in need thereof an antibody or a pharmaceutical composition thereof. The melanin antibodies may also be used for diagnostic purposes, to detect a melanoma in a sample from a subject. Also provided are methods of producing the melanin antibodies described herein.

[0049] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0050] Numeric ranges are inclusive of the numbers defining the range.

[0051] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

[0052] As used herein, the singular form "a", "an", and "the" includes plural references unless indicated otherwise.

[0053] It is understood that aspects and embodiments of the invention described herein include "comprising," "consisting," and "consisting essentially of aspects and embodiments. [0054] The term "about" as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0055] Other definitions of terms may appear throughout the specification.

[0056] For any of the structural and functional characteristics described herein, methods of determining these characteristics are known in the art.

Melanin Antibodies

[0057] Provided herein are antibodies that specifically bind to melanin. In some embodiments, the melanin is mammalian melanin, e.g. human melanin, or murine melanin. In other embodiments, the melanin is a non-mammalian melanin.

[0058] The term "antibody" as used herein throughout is in the broadest sense and includes, but is not limited to, a monoclonal antibody, polyclonal antibody, human antibody, humanized antibody, non-human antibody, chimeric antibody, bispecific antibody, multi-specific antibody, antigen- binding fragments of the antibody (e.g Fab fragment, a Fab'2 fragment, a CDR or a ScFv), antibody-drug conjugates, and other antibody fragments that retain specificity for a melanin antigen.

[0059] The antibody can be any of an IgA, IgD, IgE, IgG, or IgM antibody. The IgA antibody can be an IgAl or an IgA2 antibody. The IgG antibody can be an IgGl, IgG2, IgG2a, IgG2b, IgG3 or IgG4 antibody. A combination of any of these antibodies can also be used.

[0060] In some embodiments, the melanin antibody is conjugated for a variety of purposes including, but not limited to, for use in therapeutics, detection, diagnostics, visualization, quantification, sorting, and for use in biological assays.

[0061] In some embodiments, the antibody is a humanized antibody that specifically binds to melanin. In some embodiments, the humanized antibody is a humanized version of a mouse monoclonal 8C3 IgG antibody (NCBI GenBank accession number KX346264; Uran ME,

Nosanchuk JD, Restrepo A, Hamilton AJ, Gomez BL, Cano LE. Detection of antibodies against Paracoccidioides brasiliensis melanin in in vitro and in vivo studies during infection. Clin Vaccine Immunol. 2011 Oct; 18(10): 1680-8). [0062] In some embodiments, the antibody is a chimeric antibody that specifically binds to melanin. In an exemplary embodiment, the antibody is a chimeric mouse-human antibody. The chimeric mouse-human antibody can comprise human variable regions and mouse constant regions. In some embodiments, the constant region is of the IgG type, e.g. of the IgG type. In some embodiments, the constant region is not of the IgG type, e.g. not of the human IgG type. In some embodiments, the constant region is of the IgM type, e.g. of the human IgM type. In some embodiments, the constant region is not of the IgM type, e.g. not of the human IgM type.

[0063] Table 1 provides exemplary sequences for the antibodies and antigen-binding fragments provided herein.

Table 1; Exemplary Melanin Antibody Amino Acid Sequences

SEQ Π) NO: 1: Amino Acid Sequence of the Heavy Chain of a melanin Chimeric Antibody (8C3-hIgGl)

EVQLEES GGGL VQPGGSMK VS C AAS GFTF SD AWMD WVRQ SPEKGLEWV AEIRSKAHN

HATYYAESVKGRFTISRDDSKSSVYLQMNSLRAEDTGTYYCTRGGYYGNYGFFAYWG Q

GTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHT

FPAVLQ SSGLYSLS S WTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV FNWYVDGVEVHNAKT

KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ V

YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYS

KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ Π) NO: 2: Amino Acid Sequence of the Light Chain of a melanin Chimeric Antibody (8C3-hKappa)

ΟΙΕΜΤρβΡΑβΕΑνβΕθρΚΑΉβΟΚΑβΕβνϋβ ΥΟΤβΡΜΡΓννΥρρΚΡθρΡΡΚ ΕΙΥΕΑβΝΕΕβ GVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEYPYTFGGGTKLEIKRTVAAPSVF IFPPSDEQLKS GTAS WCLLNNF YPRE AKVQ WKVDNALQ S GNS QES VTEQD SKD S T YSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ Π) NO: 3: Amino Acid Sequence of the Heavy Chain of a melanin Humanized

Antibody (8C3-HE-VH3A-hIgGl)

EVQLVES GGGL VQPGGSMRVS C AAS GFTF SD AWMD WVRQ APGKGLEWVAEIRSKAFIN HATYYAESVKGRFTISRDDSKSTVYLQMNSLRAEDTGTYYCTRGGYYGNYGFFAYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQ SSGLYSLS S WTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP Table 1: Exemplary Melanin Antibody Amino Acid Sequences

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV FNWYVDGVEVHNAKT KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ Π ⁾ NO: 4: Amino Acid Sequence of the Heavy Chain of a melanin Humanized

Antibody (8C3-HE-VH3B-hIgGl)

EVQLVES GGGL VQPGGSMRVS C AAS GFTF SD AW1VH) WVRQ APGKGLEWVAEIRSKAFIN

HATYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTGVYYCTRGGYYGNYGFFAYWG

QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVH

TFPAVLQS SGLYSLS S WTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC

PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV FNWYVDGVEVHNAK

TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP Q

VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLY

SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ Π ⁾ NO: 5: Amino Acid Sequence of the Light Chain of a melanin Humanized Antibody (8C3-HE-VKlA-hKappa)

DIQMTQSPSSLSVSLGDRAΉTCRASESVDSYGTSFMHWYQQKPGKPPKLLIYLASNLES G VPSRFSGSGSRTDFTLTISPVQAEDFATYYCQQNNEYPYTFGQGTKLEIKRTVAAPSVFI FP PSDEQLKS GT AS WCLLNNF YPRE AKVQ WKVDNALQ S GNS QES VTEQD SKD S T YSLS S T LTLSKAD YEKHKVYACEVTHQGLS SPVTKSFNRGEC

SEQ Π ⁾ NO: 6: Amino Acid Sequence of the Light Chain of a melanin Humanized Antibody (8C3-HE-VKlB-hKappa)

DIQMTQ SPS SLS VS VGDRATITCRASES VD S YGTSFMHWYQQKPGKPPKLLI YL ASNLQ S GVPSRFSGSGSRTDFTLTISPVQAEDFATYYCQQNNEYPYTFGQGTKLEIKRTVAAPSVF I FPPSDEQLKS GT AS WCLLNNF YPRE AKVQ WKVDNALQ S GNS QES VTEQD SKD S T YSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ Π ⁾ NO: 7: Amino Acid Sequence of the Light Chain of a melanin Humanized Antibody (8C3-HE-VK4-hKappa)

DIVMTQSPDSLAVSLGERAΉNCKASESVDSYGTSFMHWYQQKPGQPPKLLIYLASNRES GWDRFSGSGSRTDFTLTISPVQAEDVATYYCQQNNEYPYTFGQGTKLEIKRTVAAPSVFI FPPSDEQLKS GT AS WCLLNNF YPRE AKVQ WKVDNALQ S GNS QES VTEQD SKD S T YSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ JD NO: 8: V _H CDR1 Table 1; Exemplary Melanin Antibody Amino Acid Sequences

FTFSDAWMD

SEQ ID NO: 9: V _H CDR2

WVAEIRSKAHNHATYY

SEQ ID NO: 10: V _H CDR3

RGGYYGNYGFFAY

SEQ ID NO: 11: V _L CDR1

ESVDSYGTSFMH

SEQ ID NO: 12: V _L CDR2

LLIYLASNLES

SEQ ID NO: 13: V _L CDR2

LLIYLASNLQS

SEQ ID NO: 14: V _L CDR2

LLIYLASNRES

SEQ ID NO: 15: V _L CDR3

QQNNEYPY

[0064] In some embodiments, the melanin antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1.

[0065] In some embodiments, the melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 2.

[0066] In some embodiments, the melanin antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2.

[0067] In some embodiments, the melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. [0068] In some embodiments, the melanin antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

[0069] In some embodiments, the melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 5.

[0070] In some embodiments, the melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 6.

[0071] In some embodiments, the melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 7.

[0072] In some embodiments, the melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 5.

[0073] In some embodiments, the melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 6.

[0074] In some embodiments, the melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 7.

[0075] In some embodiments, the melanin antibody comprises a light chain comprising the variable portion of any one of the light chain sequences provided for in Table 1. In some embodiments, the melanin antibody comprises a light chain comprising only the variable portion of any one of the light chain sequences provided for in Table 1.

[0076] In some embodiments, the melanin antibody comprises a light chain comprising the CDRs contained in any one of the light chain sequences provided for in Table 1. In some embodiments, the melanin antibody comprises a heavy chain comprising the CDRs contained in any one of the heavy chain sequences provided for in Table 1. [0077] In some embodiments, the melanin antibody comprises a heavy chain comprising the variable portion of any one of the heavy chain sequences provided for in Table 1. In some embodiments, the melanin antibody comprises a heavy chain comprising only the variable portion of any one of the heavy chain sequences provided for in Table 1.

[0078] In some embodiments, the heavy chain of the melanin antibody comprises at least one of the complementarity-determining region (CDR) sequences of SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In some embodiments, the heavy chain of the melanin antibody comprises the complementarity-determining region (CDR) sequences of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

[0079] In some embodiments, the light chain of the melanin antibody comprises at least one of the CDR sequences of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. In some embodiments, the light chain of the melanin antibody comprises the

complementarity-determining region (CDR) sequences of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 15. In some embodiments, the light chain of the melanin antibody comprises the complementarity-determining region (CDR) sequences of SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15. In some embodiments, the light chain of the melanin antibody comprises the complementarity-determining region (CDR) sequences of SEQ ID NO: 11, SEQ ID NO: 14, and SEQ ID NO: 15.

[0080] In some embodiments, the melanin antibody is a humanized antibody selected from the group consisting of HE-1, HE-2, HE-3, HE-4, HE-5, and HE-6.

[0081] In some embodiments, the melanin antibody is a bispecific antibody. For example, the bispecific antibody can comprise a first arm that targets melanin and a second arm that targets an antigen comprising an additional therapeutic target, for example an immune checkpoint inhibitor. In some embodiments, the bispecific antibody comprises a first arm that targets melanin and a second arm that targets an immune checkpoint inhibitor, for example, the second arm targets CTLA4, PD-1, or PD-Ll.

[0082] In some embodiments, the melanin antibody is conjugated to an agent including, but not limited to, a radionuclide (also referred to as a radioactive nuclide, radioisotope or radioactive isotope), a cytotoxin, a chemotherapeutic agent, a drug, an enzyme, a detectable agent, a cytokine, a hormone, an oligonucleotide, or a second antibody.

[0083] In another exemplary embodiment, the melanin antibody is conjugated to a cytotoxin.

[0084] In another exemplary embodiment, the melanin antibody is conjugated to a microtubule inhibitor.

[0085] In another exemplary embodiment, the melanin antibody is conjugated to a nucleic acid damaging agent, such as a DNA alkylator, a DNA cleaving agent, a DNA cross-linker, a DNA intercalator, or other DNA damaging agent.

[0086] In another exemplary embodiment, the melanin antibody is conjugated to a radionuclide. The choice of the particular radionuclide with which the melanin antibody is conjugated may be determined by the size of the melanoma tumor to be treated and its localization in the body, taking into consideration the emission range in the tissue and half-life. Radionuclides include alpha emitters, beta emitters, and positron emitters.

[0087] Exemplary radionuclides include but are not limited to alpha emitters, beta emitters, and positron emitters.

[0088] Examples of alpha emitters include: 213-Bismuth (half-life 46 minutes), 223-Radium (half-hfe 11.3 days), 224-Radium (half-life 3.7 days), 225-Radium (half-life 14.8 days), 225- Actinium (half life 10 days), 212-Lead (half-life 10.6 hours), 212-Bismuth (half-life 60 minutes), 211-Astatin (half-life 7.2 hours), 255-Fermium (half-life 20 hours)and 227-Thorium (half-life 18.7 days).

[0089] Examples of beta emitters include: 188-Rhenium (half-life 16.7 hours), 90- Yttrium (half- life 2.7 days), 32-Phosphorous (half-life 14.3 days), 47-Scandium (half-life 3.4 days), 67-Copper (half-life 62 hours), 64-Copper (half-life 13hours), 77-Arsenic (half-life 38.8 hours), 89-Strontium (half-hfe 51 days), 105-Rhodium (half-life 35 hours), 109-Palladium (half-life 13 hours), I l l-Silver (half-life 7.5 days), 131 Iodine (half-life 8 days), 177-Lutetium (half-life 6.7 days), 153 -Samarium (half-hfe 46.7 hours), 159- Gadolinium (half-life 18.6 hours), 186-Rhenium (half-life 3.7 days), 166- Holmium (half-life 26.8 hours), 166-Dysprosium (half-life 81.6hours), 140-Lantanum (half-life 40.3 hours), 194-Irndium (half-life 19 hours), 198-Gold (half-life 2.7 days), and 199 Gold (half-life 3.1 days). [0090] Examples of positron emitters include (half-life in parenthesis): 52Mn (21.1 min); 62Cu (9.74 mm); 68Ga (68.1 mm); 11C (20min); 82Rb (1.27 mm); 1 101η (1 .15 h); 118Sb (3.5

[0091] mm); 1221 (3.63 mm); 18F (1.83 h); 34"'C1 (32.2 mm); 38K (7.64 mm); 51Mn (46.2 mm); 52Mn (5.59 days); 52Fe (8.28 h); 55Co (17.5 h); 61Cu (3.41 h); 64Cu (12.7 h); 72As (1.08 days); 75Br (1.62 h); 76Br (16.2 h); 82"'Rb (6.47 h); 83Sr(1.35 days); 86Y (14.7 h); 89Zr (3.27 days); 94"Tc (52.0 min); 1201(1.35h); 124 1 (4.18 days). 64-Copper is a mixed positron, electron and Auger electron emitter.

[0092] Exemplary radionuclides also may include: "mTc, ²⁰¹ T1, ¹³³ Xe, ⁿ C, ⁶² Cu, ¹⁸ F, ⁶⁸ Ga, ¹³ N, ¹⁵ 0, ³⁸ K, ⁸² Rb, "«¾Tc (Technetium), ¹⁸⁸ Re, ²¹³ Bi (213-Bismuth), ¹²⁵ I, ¹³¹ 1, ⁸⁹ Zr, ^lu In, ¹²³ I, and ¹³¹ !

[0093] In some embodiments, the melanin antibody is a humanized antibody and is conjugated to ²¹³ B. In some embodiments, the melanin antibody is a humanized antibody selected from the group consisting of HE-1, HE-2, HE-3, HE-4, HE-5, and HE-6 (referring to Table 4) and is conjugated to ²¹³ B. In some embodiments, the humanized melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 and is conjugated to ²¹³ B. In some embodiments, the humanized melanin antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 and is conjugated to ²¹³ B. In some embodiments, the humanized melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 5 and is conjugated to ²¹³ B. In some embodiments, the humanized melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 6 and is conjugated to ²¹³ B. In some embodiments, the humanized melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 7 and is conjugated to ²¹³ B. In some embodiments, the humanized melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 5 and is conjugated to ²¹³ B. In some embodiments, the humanized melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 6 and is conjugated to ²¹³ B. In some embodiments, the humanized melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 4 and is conjugated to ²¹³ B. In some embodiments, the heavy chain of the humanized melanin antibody comprises at least one of the CDR sequences of SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10 and is conjugated to ²¹³ B. In some embodiments, the light chain of the humanized melanin antibody comprises at least one of the CDR sequences of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 and is conjugated to ²¹³ B.

[0094] In some embodiments, the melanin antibody is a humanized antibody and is conjugated to ¹⁷⁷ Lu. In some embodiments, the melanin antibody is a humanized antibody selected from the group consisting of HE-1, HE-2, HE-3, HE-4, HE-5, and HE-6 (referring to Table 4) and is conjugated to ¹⁷⁷ Lu. In some embodiments, the humanized melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 and is conjugated to ¹⁷⁷ Lu. In some embodiments, the humanized melanin antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 and is conjugated to ¹⁷⁷ Lu. In some embodiments, the humanized melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 5 and is conjugated to ¹⁷⁷ Lu. In some embodiments, the humanized melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 6 and is conjugated to ¹⁷⁷ Lu. In some embodiments, the humanized melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 7 and is conjugated to ¹⁷⁷ Lu. In some embodiments, the humanized melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 5 and is conjugated to ¹⁷⁷ Lu. In some embodiments, the humanized melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 6 and is conjugated to ¹⁷⁷ Lu. In some embodiments, the humanized melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 4 and is conjugated to ¹⁷⁷ Lu. In some embodiments, the heavy chain of the humanized melanin antibody comprises at least one of the CDR sequences of SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10 and is conjugated to Lu. In some embodiments, the light chain of the humanized melanin antibody comprises at least one of the CDR sequences of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 and is conjugated to ¹⁷⁷ Lu.

[0095] In different embodiments, the dose of the radionuclide in any one of the embodiments described herein for therapeutic purposes is between 1-1000 mCi.

[0096] In some embodiments, the antibody is conjugated to one or more equivalents of an agent. In some embodiments, the antibody is conjugated to one equivalent of the agent. In some embodiments, the antibody is conjugated to two, three, four, five, six, seven, eight, nine, ten, or greater than ten equivalents of the agent. In some embodiments, the mixture of antibodies is such that the average number of agents conjugated to each antibody is two, three, four, five, six, seven, eight, nine, ten, or greater than ten equivalents of the agent is one, two, three, four, five, six, seven, eight, nine, ten, or greater than ten.

[0097] In some embodiments, the antibody comprises one or more site-specific amino acid sequence modifications such that the number of agents that can be conjugated to the antibody can be modulated.

[0098] In another exemplary embodiment, the melanin antibody is conjugated to an antiinflammatory agent.

[0099] In another exemplary embodiment, the melanin antibody is conjugated to a detectable agent (label). In some embodiments, the detectable agent is a diagnostic agent. In some

embodiments, the melanin antibody is conjugated to a detectable label, a spin label, a colorimetric label, a radioactive label, an enzymatic label, a fluorescent label, or a magnetic label.

[00100] In some embodiments, the agent is conjugated to the melanin antibody via linker. In some embodiments, the agent is conjugated to the melanin antibody via a cleavable linker. In some embodiments, the agent is conjugated to the melanin antibody via a non-cleavable linker.

[00101] In some embodiments, the melanin antibody is conjugated or attached to a solid surface, for example a bead, resin or a microplate.

[00102] Provided herein are antibodies specific for melanin from any mammalian and non- mammalian species. In some embodiments, the melanin antibody is specific for human melanin. In some embodiments, the melanin antibody is cross reactive with melanin from other species. [00103] The antibodies provided herein bind melanin with specificity. In some embodiments, these antibodies bind melanin with specificity and selectivity.

[0100] In certain embodiments, an antibody provided herein has a dissociation constant (Kd) of range of O.OOOlnM to Ι μΜ. For example, Kd of the antibody may be about Ι μΜ, about 100 nM, about 50 nM, about 10 nM, about 5 nM, about 1 nM, about 0.5 nM, about 0.1 nM, about 0.05 nM, about 0.01 nM, about 0.005 nM, about 0.001 nM, about 0.0005 nM, or even about 0.0001 nM.

Production of Melanin Antibodies

[0101] A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with melanin. For example, solid-phase ELISA immunoassays may be used to select monoclonal antibodies specific to melanin (see, e.g., Harlow and Lane (1988) Antibodies, A

Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that may be used to determine specific immunoreactivity).

[0102] Production of the antibodies provided herein may be by any method known to those with skill in the art. For example, in some embodiments, the melanin antibodies are produced by recombinant cells engineered to express the desired light chains and heavy chains of the desired antibody. In some embodiments the antibodies are produced by hybridomas.

[0103] In some embodiments, any peptide comprising the melanin antigen, optionally linked to the immunogenic carrier, is used for immunization using standard protocols.

[0104] The quality and titer of generated antibodies may be assessed using techniques known to those in the art.

[0105] For the purposes of binding and expression, a signal peptide sequence may be expressed in frame with the antibody component of interest. Table 2 provides exemplary amino acid and nucleotide sequences that encode exemplary signal peptides. In some embodiments, the signal peptide assists a cell line in secretion of the antibody. In some embodiments, the signal peptide is designated "VK-I region Walker". In some embodiments the signal peptide is the native signal peptide found in many human Ig Kappa Chains. In some embodiments, the antibodies are synthesized in a cellular system and comprise a signal peptide sequence, for example the sequence of SEQ ID NO: 16. As provided herein, any one of the exemplary melanin antibody sequences provided in Table 1 may further include a signal peptide sequence. Thus in some embodiments, an antibody sequence of the invention comprises any one of SEQ ID NOs: 1-7 in combination with a N-terminal signal peptide sequence, for example the signal peptide sequence of SEQ ID NO: 16.

Table 2; Exemplary Signal Peptide Sequences

SEQ ID NO: 16: Signal peptide amino acid sequence MDMRVPAQLLGLLLLWLRGAR

SEQ ID NO: 17: Signal peptide nucleotide sequence

ATGGACATGAGAGTGCCGGCGCAACTGCTCGGCCTGCTGTTGCTGTGGCTGAGGGGA GCCAGATGC

[0106] The inventive compositions described herein also include nucleic acids encoding the antibodies, vectors comprising any of the nucleic acids encoding the antibodies, and host cells comprising any such vectors. Exemplary nucleotide sequences are provided in Table 3A. In some embodiments, the nucleic acids encoding the antibodies further include a signal peptide nucleotide sequence, for example the sequence of SEQ ID NO: 17. Table 3B provides exemplary melanin antibody expressing plasmid nucleotide sequences.

Table 3A: Exemplary Melanin Antibody Nucleotide Sequences

SEQ Π) NO: 18: DNA sequence of pABll 625.69.1 heavy chain of a chimeric melanin antibody gene (8C3-hIgGl)

GAAGTGCAGCTCGAGGAATCCGGAGGAGGACTGGTGCAGCCTGGCGGAAGCATGAAG G

TGTCATGCGCGGCTTCCGGATTCACCTTCTCGGACGCCTGGATGGATTGGGTCAGAC AAA

GCCCCGAAAAAGGCCTGGAATGGGTGGCCGAGATTCGGTCCAAGGCCCATAACCACG CC

ACCTACTACGCCGAGTCCGTGAAGGGGCGCTTTACTATCTCCCGGGATGACTCGAAG TCG

TCCGTGTACCTCCAGATGAACTCATTGAGGGCCGAGGACACTGGGACCTACTACTGT ACC

CGCGGAGGCTACTACGGGAACTATGGTTTCTTCGCCTACTGGGGCCAGGGTACCCTC GTG

ACTGTCAGCGCGGCCAGCACCAAGGGCCCCAGCGTGTTCCCACTGGCCCCAAGCTCC AA

GTCAACCTCCGGCGGAACTGCTGCGCTGGGCTGCTTGGTGAAGGACTACTTCCCCGA ACC

GGTCACCGTGTCCTGGAACAGCGGAGCCCTGACCTCGGGAGTCCACACTTTCCCCGC TGT

GCTGCAGTCGTCCGGCCTGTACTCGCTCTCGTCCGTGGTCACTGTCCCGTCCTCGTC CCTG Table 3A: Exemplary Melanin Antibody Nucleotide Sequences

GGTACTCAGACCTACATTTGCAACGTCAACCACAAGCCTTCAAACACGAAAGTGGACAA

GAAGGTCGAGCCGAAGTCCTGCGACAAAACCCATACTTGCCCTCCTTGTCCGGCTCC CGA

ACTGCTGGGCGGACCTTCCGTGTTCCTCTTCCCGCCTAAGCCGAAAGACACCCTGAT GAT

CAGCAGGACTCCGGAAGTGACATGCGTGGTGGTGGACGTGTCGCACGAGGACCCGGA GG

TCAAGTTTAATTGGTACGTGGACGGAGTGGAAGTCCACAACGCCAAGACCAAGCCAC GG

GAAGAACAGTACAATTCCACCTATCGCGTGGTGTCCGTGCTTACCGTGCTTCACCAA GAC

TGGCTGAACGGAAAGGAGTACAAGTGCAAAGTGTCAAACAAAGCCCTGCCTGCCCCA AT

CGAAAAGACCATCAGCAAGGCCAAGGGGCAGCCTCGGGAACCCCAAGTGTACACTCT CC

CGCCGTCAAGAGATGAACTGACCAAGAACCAAGTGTCCCTCACTTGTCTCGTGAAGG GA

TTCTACCCCTCCGATATCGCCGTGGAGTGGGAATCCAACGGGCAACCCGAGAACAAC TA

CAAGACCACCCCTCCGGTGCTTGATTCCGATGGCTCCTTCTTCCTCTACTCCAAGCT GACC

GTGGACAAGTCAAGATGGCAGCAGGGGAACGTGTTCTCCTGCTCCGTCATGCACGAG GC

CCTGCACAACCATTACACCCAGAAGTCTCTGTCGCTGAGCCCGGGAAAATAA

SEQ Π) NO: 19: DNA sequence of pAB2 625.48.2 light chain of a chimeric melanin antibody gene (8C3-hKappa)

GACATCCTGATGACTCAGTCACCCGCTAGCCTTGCGGTGTCCCTCGGACAACGCGCC ACC

ATCTCCTGTCGGGCCTCCGAATCCGTGGACTCCTACGGCACCTCCTTCATGCACTGG TAC

CAGCAGAAGCCAGGACAGCCTCCCAAGCTGTTGATCTATCTGGCCTCGAATCTGGAA TCA

GGAGTGCCGGCTCGGTTCAGCGGCTCCGGATCACGCACTGACTTCACGCTGACCATT GAC

CCCGTGGAGGCAGATGACGCCGCGACCTACTACTGCCAGCAGAACAACGAATACCCT TA

CACTTTCGGCGGGGGTACCAAGCTCGAAATCAAGCGGACAGTGGCAGCCCCATCGGT GT

TCATTTTCCCGCCGTCGGATGAGCAGCTCAAGTCCGGTACTGCCTCCGTGGTCTGCC TGCT

GAACAACTTTTACCCTCGCGAAGCGAAGGTCCAATGGAAAGTGGATAACGCCCTCCA GT

CCGGAAACTCCCAGGAGTCTGTCACCGAGCAGGACTCAAAGGACAGCACTTACTCCC TG

TCCTCGACTCTGACCCTGTCGAAGGCAGATTACGAGAAGCACAAAGTGTACGCCTGC GA

AGTGACCCATCAAGGCCTTTCCAGCCCGGTCACCAAGAGCTTCAATCGGGGGGAGTG T

TAG

SEQ Π) NO: 20 DNA Sequence encoding the Light Chain of a melanin Humanized

Antibody (8C3-HE-VK4-hKappa)

ATGGACATGAGAGTGCCGGCGCAACTGCTCGGCCTGCTGTTGCTGTGGCTGAGGGGA

GCCAGATGCGACATCGTGATGACTCAGTCACCCGATAGCCTTGCGGTGTCCCTCGGA

GAACGCGCCACCATCAACTGTAAAGCCTCCGAATCCGTGGACTCCTACGGCACCTCC

TTCATGCACTGGTACCAGCAGAAGCCAGGACAGCCTCCCAAGCTGTTGATCTATCTG

GCCTCGAATCGGGAATCAGGAGTGCCGGACCGGTTCAGCGGCTCCGGATCACGCACT

GACTTCACGCTGACCATTAGCCCCGTGCAAGCAGAGGACGTGGCGACCTACTACTGC

CAGCAGAACAACGAATACCCTTACACTTTCGGCCAGGGTACCAAGCTCGAAATCAAG Table 3A; Exemplary Melanin Antibody Nucleotide Sequences

CGGACAGTGGCAGCCCCATCGGTGTTCATTTTCCCGCCGTCGGATGAGCAGCTCAAG

TCCGGTACTGCCTCCGTGGTCTGCCTGCTGAACAACTTTTACCCTCGCGAAGCGAAG G

TCCAATGGAAAGTGGATAACGCCCTCCAGTCCGGAAACTCCCAGGAGTCTGTCACCG

AGCAGGACTCAAAGGACAGCACTTACTCCCTGTCCTCGACTCTGACCCTGTCGAAGG

CAGATTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCATCAAGGCCTTTCCA

GCCCGGTCACCAAGAGCTTCAATCGGGGGGAGTGTTAGTAA

SEQ Π) NO: 21 DNA Sequence encoding the Light Chain of a melanin Humanized

Antibody (8C3-HE-VKlA-hKappa)

ATGGACATGAGAGTGCCGGCGCAACTGCTCGGCCTGCTGTTGCTGTGGCTGAGGGGA

GCCAGATGCGACATCCAGATGACTCAGTCACCCTCGAGCCTTAGCGTGTCCCTCGGA

GATCGCGCCACCATCACCTGTCGGGCCTCCGAATCCGTGGACTCCTACGGCACCTCC T

TCATGCACTGGTACCAGCAGAAGCCAGGAAAGCCTCCCAAGCTGTTGATCTATCTGG

CCTCGAATCTGGAATCAGGAGTGCCGTCGCGGTTCAGCGGCTCCGGATCACGCACTG

ACTTCACGCTGACCATTAGCCCCGTGCAAGCAGAGGACTTTGCGACCTACTACTGCC

AGCAGAACAACGAATACCCTTACACTTTCGGCCAGGGTACCAAGCTCGAAATCAAGC

GGACAGTGGCAGCCCCATCGGTGTTCATTTTCCCGCCGTCGGATGAGCAGCTCAAGT

CCGGTACTGCCTCCGTGGTCTGCCTGCTGAACAACTTTTACCCTCGCGAAGCGAAGG T

CCAATGGAAAGTGGATAACGCCCTCCAGTCCGGAAACTCCCAGGAGTCTGTCACCGA

GCAGGACTCAAAGGACAGCACTTACTCCCTGTCCTCGACTCTGACCCTGTCGAAGGC

AGATTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCATCAAGGCCTTTCCAG

CCCGGTCACCAAGAGCTTCAATCGGGGGGAGTGTTAGTAA

SEQ IN NO: 22 DNA Sequence encoding Light Chain of a melanin Humanized Antibody (8C3-HE-VKlB-hKappa)

ATGGACATGAGAGTGCCGGCGCAACTGCTCGGCCTGCTGTTGCTGTGGCTGAGGGGA

GCCAGATGCGACATCCAGATGACTCAGTCACCCTCGAGCCTTAGCGTGTCCGTGGGA

GATCGCGCCACCATCACCTGTCGGGCCTCCGAATCCGTGGACTCCTACGGCACCTCC T

TCATGCACTGGTACCAGCAGAAGCCAGGAAAGCCTCCCAAGCTGTTGATCTATCTGG

CCTCGAATCTGCAGTCAGGAGTGCCGTCGCGGTTCAGCGGCTCCGGATCACGCACTG

ACTTCACGCTGACCATTAGCCCCGTGCAAGCAGAGGACTTTGCGACCTACTACTGCC

AGCAGAACAACGAATACCCTTACACTTTCGGCCAGGGTACCAAGCTCGAAATCAAGC

GGACAGTGGCAGCCCCATCGGTGTTCATTTTCCCGCCGTCGGATGAGCAGCTCAAGT

CCGGTACTGCCTCCGTGGTCTGCCTGCTGAACAACTTTTACCCTCGCGAAGCGAAGG T

CCAATGGAAAGTGGATAACGCCCTCCAGTCCGGAAACTCCCAGGAGTCTGTCACCGA

GCAGGACTCAAAGGACAGCACTTACTCCCTGTCCTCGACTCTGACCCTGTCGAAGGC

AGATTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCATCAAGGCCTTTCCAG

CCCGGTCACCAAGAGCTTCAATCGGGGGGAGTGTTAGTAA Table 3A; Exemplary Melanin Antibody Nucleotide Sequences

SEQ ID NO: 23 DNA Sequence encoding the Heavy Chain of a melanin Humanized Antibody (8C3-HE-VH3A-hIgGl)

ATGGACATGCGCGTGCCGGCACAACTGCTGGGCCTGCTGCTGCTTTGGCTGCGGGGA

GCTAGATGCGAAGTGCAGCTCGTCGAATCCGGAGGAGGACTGGTGCAGCCTGGCGG

AAGCATGCGCGTGTCATGCGCGGCTTCCGGATTCACCTTCTCGGACGCCTGGATGGA

TTGGGTCAGACAAGCGCCCGGCAAAGGCCTGGAATGGGTGGCCGAGATTCGGTCCA

AGGCCCATAACCACGCCACCTACTACGCCGAGTCCGTGAAGGGGCGCTTTACTATCT

CCCGGGATGACTCGAAGTCGACGGTGTACCTCCAGATGAACTCATTGAGGGCCGAGG

ACACTGGGACCTACTACTGTACCCGCGGAGGCTACTACGGGAACTATGGTTTCTTCG

CCTACTGGGGCCAGGGTACCCTCGTGACTGTCAGCAGCGCCAGCACCAAGGGCCCCA

GCGTGTTCCCACTGGCCCCAAGCTCCAAGTCAACCTCCGGCGGAACTGCTGCGCTGG

GCTGCTTGGTGAAGGACTACTTCCCCGAACCGGTCACCGTGTCCTGGAACAGCGGAG

CCCTGACCTCGGGAGTCCACACTTTCCCCGCTGTGCTGCAGTCGTCCGGCCTGTACT C

GCTCTCGTCCGTGGTCACTGTCCCGTCCTCGTCCCTGGGTACTCAGACCTACATTTG C

AACGTCAACCACAAGCCTTCAAACACGAAAGTGGACAAGAAGGTCGAGCCGAAGTC

CTGCGACAAAACCCATACTTGCCCTCCTTGTCCGGCTCCCGAACTGCTGGGCGGACC T

TCCGTGTTCCTCTTCCCGCCTAAGCCGAAAGACACCCTGATGATCAGCAGGACTCCG

GAAGTGACATGCGTGGTGGTGGACGTGTCGCACGAGGACCCGGAGGTCAAGTTTAAT

TGGTACGTGGACGGAGTGGAAGTCCACAACGCCAAGACCAAGCCACGGGAAGAACA

GTACAATTCCACCTATCGCGTGGTGTCCGTGCTTACCGTGCTTCACCAAGACTGGCT G

AACGGAAAGGAGTACAAGTGCAAAGTGTCAAACAAAGCCCTGCCTGCCCCAATCGA

AAAGACCATCAGCAAGGCCAAGGGGCAGCCTCGGGAACCCCAAGTGTACACTCTCC

CGCCGTCAAGAGATGAACTGACCAAGAACCAAGTGTCCCTCACTTGTCTCGTGAAGG

GATTCTACCCCTCCGATATCGCCGTGGAGTGGGAATCCAACGGGCAACCCGAGAACA

ACTACAAGACCACCCCTCCGGTGCTTGATTCCGATGGCTCCTTCTTCCTCTACTCCA A

GCTGACCGTGGACAAGTCAAGATGGCAGCAGGGGAACGTGTTCTCCTGCTCCGTCAT

GCACGAGGCCCTGCACAACCATTACACCCAGAAGTCTCTGTCGCTGAGCCCGGGAAA

ATAA

SEQ Π) NO: 24 DNA Sequence encoding the Heavy Chain of a melanin Humanized Antibody (8C3-HE-VH3B-hIgGl)

ATGGACATGCGCGTGCCGGCACAACTGCTGGGCCTGCTGCTGCTTTGGCTGCGGGGA

GCTAGATGCGAAGTGCAGCTCGTGGAATCCGGAGGAGGACTGGTGCAGCCTGGCGG

AAGCATGCGCGTGTCATGCGCGGCTTCCGGATTCACCTTCTCGGACGCCTGGATGGA

TTGGGTCAGACAAGCGCCCGGCAAAGGCCTGGAATGGGTGGCCGAGATTCGGTCCA

AGGCCCATAACCACGCCACCTACTACGCCGACTCCGTGAAGGGGCGCTTTACTATCT

CCCGGGATAACTCGAAGAATACCGTGTACCTCCAGATGAACTCATTGAGGGCCGAGG

ACACTGGGGTCTACTACTGTACCCGCGGAGGCTACTACGGGAACTATGGTTTCTTCG Table 3A; Exemplary Melanin Antibody Nucleotide Sequences

CCTACTGGGGCCAGGGTACCCTCGTGACTGTCAGCAGCGCCAGCACCAAGGGCCCCA

GCGTGTTCCCACTGGCCCCAAGCTCCAAGTCAACCTCCGGCGGAACTGCTGCGCTGG

GCTGCTTGGTGAAGGACTACTTCCCCGAACCGGTCACCGTGTCCTGGAACAGCGGAG

CCCTGACCTCGGGAGTCCACACTTTCCCCGCTGTGCTGCAGTCGTCCGGCCTGTACT C

GCTCTCGTCCGTGGTCACTGTCCCGTCCTCGTCCCTGGGTACTCAGACCTACATTTG C

AACGTCAACCACAAGCCTTCAAACACGAAAGTGGACAAGAAGGTCGAGCCGAAGTC

CTGCGACAAAACCCATACTTGCCCTCCTTGTCCGGCTCCCGAACTGCTGGGCGGACC T

TCCGTGTTCCTCTTCCCGCCTAAGCCGAAAGACACCCTGATGATCAGCAGGACTCCG

GAAGTGACATGCGTGGTGGTGGACGTGTCGCACGAGGACCCGGAGGTCAAGTTTAAT

TGGTACGTGGACGGAGTGGAAGTCCACAACGCCAAGACCAAGCCACGGGAAGAACA

GTACAATTCCACCTATCGCGTGGTGTCCGTGCTTACCGTGCTTCACCAAGACTGGCT G

AACGGAAAGGAGTACAAGTGCAAAGTGTCAAACAAAGCCCTGCCTGCCCCAATCGA

AAAGACCATCAGCAAGGCCAAGGGGCAGCCTCGGGAACCCCAAGTGTACACTCTCC

CGCCGTCAAGAGATGAACTGACCAAGAACCAAGTGTCCCTCACTTGTCTCGTGAAGG

GATTCTACCCCTCCGATATCGCCGTGGAGTGGGAATCCAACGGGCAACCCGAGAACA

ACTACAAGACCACCCCTCCGGTGCTTGATTCCGATGGCTCCTTCTTCCTCTACTCCA A

GCTGACCGTGGACAAGTCAAGATGGCAGCAGGGGAACGTGTTCTCCTGCTCCGTCAT

GCACGAGGCCCTGCACAACCATTACACCCAGAAGTCTCTGTCGCTGAGCCCGGGAAA

ATAA

Table 3B; Exemplary Melanin Antibody Expressing Plasmid Nucleotide Sequences

SEQ ID NO: 25 DNA Sequence of a plasmid encoding the Light Chain of a melanin Humanized Antibody (8C3-HE-VK4-hKappa)

TGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCT

TTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATT T

GAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTG

CCACCTGGGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTT A

AATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAA

AGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATT

AAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCC

CACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCAC

TAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCG

AACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGG

CAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGC

TACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGG

TGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGAT

TAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTG

AGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGG

TCGACGGTATCGATAAGCTTGATATCGAATTCGCTGGGCTGAGACCCGCAGAGGAAG

ACGCTCTAGGGATTTGTCCCGGACTAGCGAGATGGCAAGGCTGAGGACGGGAGGCT

GATTGAGAGGCGAAGGTACACCCTAATCTCAATACAACCCTTGGAGCTAAGCCAGCA

ATGGTAGAGGGAAGATTCTGCACGTCCCTTCCAGGCGGCCTCCCCGTCACCACCCAC

CCCAACCCGCCCCGACCGGAGCTGAGAGTAATTCATACAAAAGGACTCGCCCCTGCC

TTGGGGAATCCCAGGGACCGTCGTTAAACTCCCACTAACGTAGAACCCAGAGATCGC

TGCGTTCCCGCCCCCTCACCCGCCCGCTCTCGTCATCACTGAGGTGGAGAAGAGCAT

GCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAG

AAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGT

AAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGA

ACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGC C

AGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTAT

GGCCCTTGCGTGCCTTGAATTACTTCCACGCCCCTGGCTGCAGTACGTGATTCTTGA T

CCCGAGCTTCGGGTTGAAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCC

CCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCTTGGGCGCTGGGGCCGCCGCGTGCGA

ATCTGGTGGCACCTTCGCGCCTATCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAA A

ATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGG G

CCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCG

TGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAA

TCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGC

CGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAG

CGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGG Table 3B; Exemplary Melanin Antibody Expressing Plasmid Nucleotide Sequences

CGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTC

CTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGA

TTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGC G

ATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTG

ATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAG CC

TCAGACAGTGGTTCAAAGTTTTTTCCTTCCATTTCAGGTGTCGTGAAAACTACCCCT A

AAAGCCAAATCTAGAGCCACCATGGACATGAGAGTGCCGGCGCAACTGCTCGGCCT

GCTGTTGCTGTGGCTGAGGGGAGCCAGATGCGACATCGTGATGACTCAGTCACCCGA

TAGCCTTGCGGTGTCCCTCGGAGAACGCGCCACCATCAACTGTAAAGCCTCCGAATC

CGTGGACTCCTACGGCACCTCCTTCATGCACTGGTACCAGCAGAAGCCAGGACAGCC

TCCCAAGCTGTTGATCTATCTGGCCTCGAATCGGGAATCAGGAGTGCCGGACCGGTT

CAGCGGCTCCGGATCACGCACTGACTTCACGCTGACCATTAGCCCCGTGCAAGCAGA

GGACGTGGCGACCTACTACTGCCAGCAGAACAACGAATACCCTTACACTTTCGGCCA

GGGTACCAAGCTCGAAATCAAGCGGACAGTGGCAGCCCCATCGGTGTTCATTTTCCC

GCCGTCGGATGAGCAGCTCAAGTCCGGTACTGCCTCCGTGGTCTGCCTGCTGAACAA

CTTTTACCCTCGCGAAGCGAAGGTCCAATGGAAAGTGGATAACGCCCTCCAGTCCGG

AAACTCCCAGGAGTCTGTCACCGAGCAGGACTCAAAGGACAGCACTTACTCCCTGTC

CTCGACTCTGACCCTGTCGAAGGCAGATTACGAGAAGCACAAAGTGTACGCCTGCGA

AGTGACCCATCAAGGCCTTTCCAGCCCGGTCACCAAGAGCTTCAATCGGGGGGAGTG

TTAGTAATGAGGATCCCCCTATTCTATAGTGTCACCTAAATGCTAGAGCTCGCTGAT C

AGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCC TT

CCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTG C

ATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAG

CAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTA

TGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGAGCGGCCGCCCCTTCTGAGGCG

GAAAGAACCAGCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCC

CAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGA

AAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCA

GCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCC G

CCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCG C

CTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTT T

TGCAAAAAAGCTAGCTTCCCGCTGCCATCATGGTTCGACCATTGAACTGCATCGTCG

CCGTGTCCCAAAATATGGGGATTGGCAAGAACGGAGACCTACCCTGGCCTCCGCTCA

GGAACGAGTTCAAGTACTTCCAAAGAATGACCACAACCTCTTCAGTGGAAGGTAAAC

AGAATCTGGTGATTATGGGTAGGAAAACCTGGTTCTCCATTCCTGAGAAGAATCGAC

CTTTAAAGGACAGAATTAATATAGTTCTCAGTAGAGAACTCAAAGAACCACCACGAG

GAGCTCATTTTCTTGCCAAAAGTTTGGATGATGCCTTAAGACTTATTGAACAACCGG A

ATTGGCAAGTAAAGTAGACATGGTTTGGATAGTCGGAGGCAGTTCTGTTTACCAGGA

AGCCATGAATCAACCAGGCCACCTTAGACTCTTTGTGACAAGGATCATGCAGGAATT

TGAAAGTGACACGTTTTTCCCAGAAATTGATTTGGGGAAATATAAACTTCTCCCAGA

ATACCCAGGCGTCCTCTCTGAGGTCCAGGAGGAAAAAGGCATCAAGTATAAGTTTGA

AGTCTACGAGAAGAAAGACTAACAGGAAGATGCTTTCAAGTTCTCTGCTCCCCTCCT Table 3B; Exemplary Melanin Antibody Expressing Plasmid Nucleotide Sequences

AAAGCTATGCATTTTTATAAGACCATGGGACTTTTGCTGGCTTTAGATCCCGCGGAGA

TCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTG

AAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTAT A

AGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAG

GGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCT

GATTATGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTA A

TCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAAC A

TACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTC

ACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAG C

TGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTT

CCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTAT C

AGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAA

AGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTG

CTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCA

AGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGG

AAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGC C

TTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGT T

CGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCG

ACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACT T

ATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCG

GTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTAT

TTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTT G

ATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGAT

TACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGA

CGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAG

GATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTAT A

TATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCA

GCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACT A

CGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCAC

GCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGC

AGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAA G

CTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAG G

CATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACG A

TCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGT

CCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCA G

CACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTG A

GTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCC

GGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCAT

TGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAG

TTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAG C

GTTTCTGGGTGAGCAAAAACAGGAAGGCAAAA Table 3B; Exemplary Melanin Antibody Expressing Plasmid Nucleotide Sequences

SEQ ID NO: 26 DNA Sequence of a plasmid encoding the Light Chain of a melanin

Humanized Antibody (8C3-HE-VKlA-hKappa)

CGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGC

GATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCA

GTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCG

AGGTCGACGGTATCGATAAGCTTGATATCGAATTCGCTGGGCTGAGACCCGCAGAGG

AAGACGCTCTAGGGATTTGTCCCGGACTAGCGAGATGGCAAGGCTGAGGACGGGAG

GCTGATTGAGAGGCGAAGGTACACCCTAATCTCAATACAACCCTTGGAGCTAAGCCA

GCAATGGTAGAGGGAAGATTCTGCACGTCCCTTCCAGGCGGCCTCCCCGTCACCACC

CACCCCAACCCGCCCCGACCGGAGCTGAGAGTAATTCATACAAAAGGACTCGCCCCT

GCCTTGGGGAATCCCAGGGACCGTCGTTAAACTCCCACTAACGTAGAACCCAGAGAT

CGCTGCGTTCCCGCCCCCTCACCCGCCCGCTCTCGTCATCACTGAGGTGGAGAAGAG

CATGCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCC

GAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGG

GGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGG

AGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGC C

GCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGT

TATGGCCCTTGCGTGCCTTGAATTACTTCCACGCCCCTGGCTGCAGTACGTGATTCT T

GATCCCGAGCTTCGGGTTGAAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGA

GCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCTTGGGCGCTGGGGCCGCCGCGTG

CGAATCTGGTGGCACCTTCGCGCCTATCTCGCTGCTTTCGATAAGTCTCTAGCCATT T

AAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATG C

GGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGC

CCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGA

GAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGC

CGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGT

GAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACG

CGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCC

GTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCT

CGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTA T

GCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCAC

TTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTC AA

GCCTCAGACAGTGGTTCAAAGTTTTTTCCTTCCATTTCAGGTGTCGTGAAAACTACC C

CTAAAAGCCAAATCTAGAGCCACCATGGACATGAGAGTGCCGGCGCAACTGCTCGG

CCTGCTGTTGCTGTGGCTGAGGGGAGCCAGATGCGACATCCAGATGACTCAGTCACC

CTCGAGCCTTAGCGTGTCCCTCGGAGATCGCGCCACCATCACCTGTCGGGCCTCCGA

ATCCGTGGACTCCTACGGCACCTCCTTCATGCACTGGTACCAGCAGAAGCCAGGAAA

GCCTCCCAAGCTGTTGATCTATCTGGCCTCGAATCTGGAATCAGGAGTGCCGTCGCG

GTTCAGCGGCTCCGGATCACGCACTGACTTCACGCTGACCATTAGCCCCGTGCAAGC Table 3B; Exemplary Melanin Antibody Expressing Plasmid Nucleotide Sequences

AGAGGACTTTGCGACCTACTACTGCCAGCAGAACAACGAATACCCTTACACTTTCGG

CCAGGGTACCAAGCTCGAAATCAAGCGGACAGTGGCAGCCCCATCGGTGTTCATTTT

CCCGCCGTCGGATGAGCAGCTCAAGTCCGGTACTGCCTCCGTGGTCTGCCTGCTGAA

CAACTTTTACCCTCGCGAAGCGAAGGTCCAATGGAAAGTGGATAACGCCCTCCAGTC

CGGAAACTCCCAGGAGTCTGTCACCGAGCAGGACTCAAAGGACAGCACTTACTCCCT

GTCCTCGACTCTGACCCTGTCGAAGGCAGATTACGAGAAGCACAAAGTGTACGCCTG

CGAAGTGACCCATCAAGGCCTTTCCAGCCCGGTCACCAAGAGCTTCAATCGGGGGGA

GTGTTAGTAATGAGGATCCCCCTATTCTATAGTGTCACCTAAATGCTAGAGCTCGCT G

ATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGT G

CCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAA A

TTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGG

ACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGC

TCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGAGCGGCCGCCCCTTCTGA

GGCGGAAAGAACCAGCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGG

CTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTG

TGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTA

GTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAG T

TCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAG G

CCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAG

GCTTTTGCAAAAAAGCTAGCTTCCCGCTGCCATCATGGTTCGACCATTGAACTGCAT C

GTCGCCGTGTCCCAAAATATGGGGATTGGCAAGAACGGAGACCTACCCTGGCCTCCG

CTCAGGAACGAGTTCAAGTACTTCCAAAGAATGACCACAACCTCTTCAGTGGAAGGT

AAACAGAATCTGGTGATTATGGGTAGGAAAACCTGGTTCTCCATTCCTGAGAAGAAT

CGACCTTTAAAGGACAGAATTAATATAGTTCTCAGTAGAGAACTCAAAGAACCACCA

CGAGGAGCTCATTTTCTTGCCAAAAGTTTGGATGATGCCTTAAGACTTATTGAACAA C

CGGAATTGGCAAGTAAAGTAGACATGGTTTGGATAGTCGGAGGCAGTTCTGTTTACC

AGGAAGCCATGAATCAACCAGGCCACCTTAGACTCTTTGTGACAAGGATCATGCAGG

AATTTGAAAGTGACACGTTTTTCCCAGAAATTGATTTGGGGAAATATAAACTTCTCC C

AGAATACCCAGGCGTCCTCTCTGAGGTCCAGGAGGAAAAAGGCATCAAGTATAAGTT

TGAAGTCTACGAGAAGAAAGACTAACAGGAAGATGCTTTCAAGTTCTCTGCTCCCCT

CCTAAAGCTATGCATTTTTATAAGACCATGGGACTTTTGCTGGCTTTAGATCCCGCG G

AGATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCA

GTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCAT T

ATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTT C

AGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGG

CTGATTATGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCG T

AATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACA A

CATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAAC

TCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCC A

GCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTC

TTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGT A

TCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGG Table 3B; Exemplary Melanin Antibody Expressing Plasmid Nucleotide Sequences

AAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCG

TTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGC T

CAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTG

GAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCG C

CTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAG T

TCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCC

GACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGAC

TTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGC

GGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTA

TTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCT T

GATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGA

TTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTG

ACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAA

GGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTA T

ATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTC

AGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAAC T

ACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCA

CGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGC

AGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAA G

CTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAG G

CATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACG A

TCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGT

CCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCA G

CACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTG A

GTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCC

GGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCAT

TGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAG

TTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAG C

GTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGC

GACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTA T

CAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAA

ATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGGGAAATTGTAAACGTTA

ATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAAT AG

GCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAG

TGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAA

AGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATC

AAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCC

CCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGA

AAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTA

ACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTC

AGGCTGCGCAACTGTTGGGAAGGGCGAT Table 3B; Exemplary Melanin Antibody Expressing Plasmid Nucleotide Sequences

SEQ ID NO: 27 DNA Sequence of a plasmid encoding the Light Chain of a melanin

Humanized Antibody (8C3-HE-VKlB-hKappa)

CGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGC

GATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCA

GTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCG

AGGTCGACGGTATCGATAAGCTTGATATCGAATTCGCTGGGCTGAGACCCGCAGAGG

AAGACGCTCTAGGGATTTGTCCCGGACTAGCGAGATGGCAAGGCTGAGGACGGGAG

GCTGATTGAGAGGCGAAGGTACACCCTAATCTCAATACAACCCTTGGAGCTAAGCCA

GCAATGGTAGAGGGAAGATTCTGCACGTCCCTTCCAGGCGGCCTCCCCGTCACCACC

CACCCCAACCCGCCCCGACCGGAGCTGAGAGTAATTCATACAAAAGGACTCGCCCCT

GCCTTGGGGAATCCCAGGGACCGTCGTTAAACTCCCACTAACGTAGAACCCAGAGAT

CGCTGCGTTCCCGCCCCCTCACCCGCCCGCTCTCGTCATCACTGAGGTGGAGAAGAG

CATGCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCC

GAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGG

GGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGG

AGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGC C

GCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGT

TATGGCCCTTGCGTGCCTTGAATTACTTCCACGCCCCTGGCTGCAGTACGTGATTCT T

GATCCCGAGCTTCGGGTTGAAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGA

GCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCTTGGGCGCTGGGGCCGCCGCGTG

CGAATCTGGTGGCACCTTCGCGCCTATCTCGCTGCTTTCGATAAGTCTCTAGCCATT T

AAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATG C

GGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGC

CCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGA

GAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGC

CGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGT

GAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACG

CGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCC

GTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCT

CGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTA T

GCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCAC

TTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTC AA

GCCTCAGACAGTGGTTCAAAGTTTTTTCCTTCCATTTCAGGTGTCGTGAAAACTACC C

CTAAAAGCCAAATCTAGAGCCACCATGGACATGAGAGTGCCGGCGCAACTGCTCGG

CCTGCTGTTGCTGTGGCTGAGGGGAGCCAGATGCGACATCCAGATGACTCAGTCACC

CTCGAGCCTTAGCGTGTCCGTGGGAGATCGCGCCACCATCACCTGTCGGGCCTCCGA

ATCCGTGGACTCCTACGGCACCTCCTTCATGCACTGGTACCAGCAGAAGCCAGGAAA

GCCTCCCAAGCTGTTGATCTATCTGGCCTCGAATCTGCAGTCAGGAGTGCCGTCGCG

GTTCAGCGGCTCCGGATCACGCACTGACTTCACGCTGACCATTAGCCCCGTGCAAGC Table 3B; Exemplary Melanin Antibody Expressing Plasmid Nucleotide Sequences

AGAGGACTTTGCGACCTACTACTGCCAGCAGAACAACGAATACCCTTACACTTTCGG

CCAGGGTACCAAGCTCGAAATCAAGCGGACAGTGGCAGCCCCATCGGTGTTCATTTT

CCCGCCGTCGGATGAGCAGCTCAAGTCCGGTACTGCCTCCGTGGTCTGCCTGCTGAA

CAACTTTTACCCTCGCGAAGCGAAGGTCCAATGGAAAGTGGATAACGCCCTCCAGTC

CGGAAACTCCCAGGAGTCTGTCACCGAGCAGGACTCAAAGGACAGCACTTACTCCCT

GTCCTCGACTCTGACCCTGTCGAAGGCAGATTACGAGAAGCACAAAGTGTACGCCTG

CGAAGTGACCCATCAAGGCCTTTCCAGCCCGGTCACCAAGAGCTTCAATCGGGGGGA

GTGTTAGTAATGAGGATCCCCCTATTCTATAGTGTCACCTAAATGCTAGAGCTCGCT G

ATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGT G

CCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAA A

TTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGG

ACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGC

TCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGAGCGGCCGCCCCTTCTGA

GGCGGAAAGAACCAGCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGG

CTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTG

TGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTA

GTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAG T

TCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAG G

CCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAG

GCTTTTGCAAAAAAGCTAGCTTCCCGCTGCCATCATGGTTCGACCATTGAACTGCAT C

GTCGCCGTGTCCCAAAATATGGGGATTGGCAAGAACGGAGACCTACCCTGGCCTCCG

CTCAGGAACGAGTTCAAGTACTTCCAAAGAATGACCACAACCTCTTCAGTGGAAGGT

AAACAGAATCTGGTGATTATGGGTAGGAAAACCTGGTTCTCCATTCCTGAGAAGAAT

CGACCTTTAAAGGACAGAATTAATATAGTTCTCAGTAGAGAACTCAAAGAACCACCA

CGAGGAGCTCATTTTCTTGCCAAAAGTTTGGATGATGCCTTAAGACTTATTGAACAA C

CGGAATTGGCAAGTAAAGTAGACATGGTTTGGATAGTCGGAGGCAGTTCTGTTTACC

AGGAAGCCATGAATCAACCAGGCCACCTTAGACTCTTTGTGACAAGGATCATGCAGG

AATTTGAAAGTGACACGTTTTTCCCAGAAATTGATTTGGGGAAATATAAACTTCTCC C

AGAATACCCAGGCGTCCTCTCTGAGGTCCAGGAGGAAAAAGGCATCAAGTATAAGTT

TGAAGTCTACGAGAAGAAAGACTAACAGGAAGATGCTTTCAAGTTCTCTGCTCCCCT

CCTAAAGCTATGCATTTTTATAAGACCATGGGACTTTTGCTGGCTTTAGATCCCGCG G

AGATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCA

GTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCAT T

ATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTT C

AGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGG

CTGATTATGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCG T

AATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACA A

CATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAAC

TCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCC A

GCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTC

TTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGT A

TCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGG Table 3B; Exemplary Melanin Antibody Expressing Plasmid Nucleotide Sequences

AAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCG

TTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGC T

CAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTG

GAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCG C

CTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAG T

TCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCC

GACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGAC

TTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGC

GGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTA

TTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCT T

GATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGA

TTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTG

ACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAA

GGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTA T

ATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTC

AGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAAC T

ACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCA

CGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGC

AGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAA G

CTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAG G

CATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACG A

TCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGT

CCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCA G

CACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTG A

GTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCC

GGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCAT

TGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAG

TTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAG C

GTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGC

GACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTA T

CAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAA

ATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGGGAAATTGTAAACGTTA

ATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAAT AG

GCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAG

TGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAA

AGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATC

AAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCC

CCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGA

AAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTA

ACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTC

AGGCTGCGCAACTGTTGGGAAGGGCGAT Table 3B; Exemplary Melanin Antibody Expressing Plasmid Nucleotide Sequences

SEQ ID NO: 28 DNA Sequence of a plasmid encoding the Heavy Chain of a melanin Humanized Antibody (8C3-HE-VH3A-hIgGl

CGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGC

GATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCA

GTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCG

AGGTCGACGGTATCGATAAGCTTGATATCGAATTCGCTGGGCTGAGACCCGCAGAGG

AAGACGCTCTAGGGATTTGTCCCGGACTAGCGAGATGGCAAGGCTGAGGACGGGAG

GCTGATTGAGAGGCGAAGGTACACCCTAATCTCAATACAACCCTTGGAGCTAAGCCA

GCAATGGTAGAGGGAAGATTCTGCACGTCCCTTCCAGGCGGCCTCCCCGTCACCACC

CACCCCAACCCGCCCCGACCGGAGCTGAGAGTAATTCATACAAAAGGACTCGCCCCT

GCCTTGGGGAATCCCAGGGACCGTCGTTAAACTCCCACTAACGTAGAACCCAGAGAT

CGCTGCGTTCCCGCCCCCTCACCCGCCCGCTCTCGTCATCACTGAGGTGGAGAAGAG

CATGCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCC

GAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGG

GGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGG

AGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGC C

GCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGT

TATGGCCCTTGCGTGCCTTGAATTACTTCCACGCCCCTGGCTGCAGTACGTGATTCT T

GATCCCGAGCTTCGGGTTGAAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGA

GCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCTTGGGCGCTGGGGCCGCCGCGTG

CGAATCTGGTGGCACCTTCGCGCCTATCTCGCTGCTTTCGATAAGTCTCTAGCCATT T

AAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATG C

GGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGC

CCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGA

GAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGC

CGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGT

GAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACG

CGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCC

GTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCT

CGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTA T

GCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCAC

TTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTC AA

GCCTCAGACAGTGGTTCAAAGTTTTTTCCTTCCATTTCAGGTGTCGTGAAAACTACC C

CTAAAAGCCAAATCTAGAGCCACCATGGACATGCGCGTGCCGGCACAACTGCTGGGC

CTGCTGCTGCTTTGGCTGCGGGGAGCTAGATGCGAAGTGCAGCTCGTCGAATCCGGA

GGAGGACTGGTGCAGCCTGGCGGAAGCATGCGCGTGTCATGCGCGGCTTCCGGATTC

ACCTTCTCGGACGCCTGGATGGATTGGGTCAGACAAGCGCCCGGCAAAGGCCTGGAA

TGGGTGGCCGAGATTCGGTCCAAGGCCCATAACCACGCCACCTACTACGCCGAGTCC

GTGAAGGGGCGCTTTACTATCTCCCGGGATGACTCGAAGTCGACGGTGTACCTCCAG Table 3B; Exemplary Melanin Antibody Expressing Plasmid Nucleotide Sequences

ATGAACTCATTGAG GCCGAG }ACACTGGGACCTACTACTGTACCCGCGGAGGCTAC

TACGGGAACTATGGTTTCTTCGCCTACTGGGGCCAGGGTACCCTCGTGACTGTCAGC

AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCACTGGCCCCAAGCTCCAAGTCAACC

TCCGGCGGAACTGCTGCGCTGGGCTGCTTGGTGAAGGACTACTTCCCCGAACCGGTC

ACCGTGTCCTGGAACAGCGGAGCCCTGACCTCGGGAGTCCACACTTTCCCCGCTGTG

CTGCAGTCGTCCGGCCTGTACTCGCTCTCGTCCGTGGTCACTGTCCCGTCCTCGTCC C

TGGGTACTCAGACCTACATTTGCAACGTCAACCACAAGCCTTCAAACACGAAAGTGG

ACAAGAAGGTCGAGCCGAAGTCCTGCGACAAAACCCATACTTGCCCTCCTTGTCCGG

CTCCCGAACTGCTGGGCGGACCTTCCGTGTTCCTCTTCCCGCCTAAGCCGAAAGACA C

CCTGATGATCAGCAGGACTCCGGAAGTGACATGCGTGGTGGTGGACGTGTCGCACGA

GGACCCGGAGGTCAAGTTTAATTGGTACGTGGACGGAGTGGAAGTCCACAACGCCA

AGACCAAGCCACGGGAAGAACAGTACAATTCCACCTATCGCGTGGTGTCCGTGCTTA

CCGTGCTTCACCAAGACTGGCTGAACGGAAAGGAGTACAAGTGCAAAGTGTCAAAC

AAAGCCCTGCCTGCCCCAATCGAAAAGACCATCAGCAAGGCCAAGGGGCAGCCTCG

GGAACCCCAAGTGTACACTCTCCCGCCGTCAAGAGATGAACTGACCAAGAACCAAGT

GTCCCTCACTTGTCTCGTGAAGGGATTCTACCCCTCCGATATCGCCGTGGAGTGGGA A

TCCAACGGGCAACCCGAGAACAACTACAAGACCACCCCTCCGGTGCTTGATTCCGAT

GGCTCCTTCTTCCTCTACTCCAAGCTGACCGTGGACAAGTCAAGATGGCAGCAGGGG

AACGTGTTCTCCTGCTCCGTCATGCACGAGGCCCTGCACAACCATTACACCCAGAAG

TCTCTGTCGCTGAGCCCGGGAAAATAATGAGGATCCCCCTATTCTATAGTGTCACCT A

AATGCTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTT G

TTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTT CC

TAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGG

GGTGGGGTGGGGC A GG A C A GC A A GGGGG A GG A TTGGG A A G A C A A T A GC A GGC A TG

CTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGA

GCGGCCGCAGATTGTACCTTCTGAGGCGGAAAGAACCAGCTGTGGAATGTGTGTCAG

TTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCAT

CTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGT

ATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCC

ATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATT T

TTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGT G

AGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTACCATGATTGAACAAGA

TGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTG

GGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGG

GCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGA

CGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCT

CGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGC

AGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATG C

AATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAA

ACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGA

TCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGC

GCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAA Table 3B; Exemplary Melanin Antibody Expressing Plasmid Nucleotide Sequences

TATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTG

GCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGC

GGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAG C

GCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGGGATCGCGGAGATCCAGA C

ATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAA

ATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTG CA

ATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGG

TGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATG

AGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGG T

CATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAG C

CGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAAT

TGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTA A

TGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCC

TCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCAC T

CAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATG

TGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTT

TTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAG

GTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCT

CGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCC TT

CGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGG T

CGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGC

CTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACT G

GCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGA

GTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTG

CGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAA

ACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAG

AAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTG

GAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCAC

CTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTA A

ACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGT C

TATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGG A

GGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGC

TCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTC

CTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAA G

TAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGT G

TCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGA G

TTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCG T

TGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAA

TTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAAC C

AAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATA

CGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGT

TCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAA Table 3B; Exemplary Melanin Antibody Expressing Plasmid Nucleotide Sequences

CCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGT

GAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAA

ATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTA TT

GTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTC

CGCGCACATTTCCCCGAAAAGTGCCACCTGGGAAATTGTAAACGTTAATATTTTGTT A

AAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATC G

GCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAG

TTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAA

ACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTG G

GGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGA

GCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAG

GAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACA

CCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGC

AACTGTTGGGAAGGGCGAT

SEQ ID NO: 29 DNA Sequence of a plasmid encoding the Heavy Chain of a melanin Humanized Antibody (8C3-HE-VH3B-hIgGl)

CGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGC

GATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCA

GTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCG

AGGTCGACGGTATCGATAAGCTTGATATCGAATTCGCTGGGCTGAGACCCGCAGAGG

AAGACGCTCTAGGGATTTGTCCCGGACTAGCGAGATGGCAAGGCTGAGGACGGGAG

GCTGATTGAGAGGCGAAGGTACACCCTAATCTCAATACAACCCTTGGAGCTAAGCCA

GCAATGGTAGAGGGAAGATTCTGCACGTCCCTTCCAGGCGGCCTCCCCGTCACCACC

CACCCCAACCCGCCCCGACCGGAGCTGAGAGTAATTCATACAAAAGGACTCGCCCCT

GCCTTGGGGAATCCCAGGGACCGTCGTTAAACTCCCACTAACGTAGAACCCAGAGAT

CGCTGCGTTCCCGCCCCCTCACCCGCCCGCTCTCGTCATCACTGAGGTGGAGAAGAG

CATGCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCC

GAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGG

GGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGG

AGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGC C

GCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGT

TATGGCCCTTGCGTGCCTTGAATTACTTCCACGCCCCTGGCTGCAGTACGTGATTCT T

GATCCCGAGCTTCGGGTTGAAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGA

GCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCTTGGGCGCTGGGGCCGCCGCGTG

CGAATCTGGTGGCACCTTCGCGCCTATCTCGCTGCTTTCGATAAGTCTCTAGCCATT T

AAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATG C

GGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGC

CCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGA

GAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGC Table 3B; Exemplary Melanin Antibody Expressing Plasmid Nucleotide Sequences

CGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGT

GAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACG

CGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCC

GTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCT

CGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTA T

GCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCAC

TTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTC AA

GCCTCAGACAGTGGTTCAAAGTTTTTTCCTTCCATTTCAGGTGTCGTGAAAACTACC C

CTAAAAGCCAAATCTAGAGCCACCATGGACATGCGCGTGCCGGCACAACTGCTGGGC

CTGCTGCTGCTTTGGCTGCGGGGAGCTAGATGCGAAGTGCAGCTCGTGGAATCCGGA

GGAGGACTGGTGCAGCCTGGCGGAAGCATGCGCGTGTCATGCGCGGCTTCCGGATTC

ACCTTCTCGGACGCCTGGATGGATTGGGTCAGACAAGCGCCCGGCAAAGGCCTGGAA

TGGGTGGCCGAGATTCGGTCCAAGGCCCATAACCACGCCACCTACTACGCCGACTCC

GTGAAGGGGCGCTTTACTATCTCCCGGGATAACTCGAAGAATACCGTGTACCTCCAG

ATGAACTCATTGAGGGCCGAGGACACTGGGGTCTACTACTGTACCCGCGGAGGCTAC

TACGGGAACTATGGTTTCTTCGCCTACTGGGGCCAGGGTACCCTCGTGACTGTCAGC

AGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCACTGGCCCCAAGCTCCAAGTCAACC

TCCGGCGGAACTGCTGCGCTGGGCTGCTTGGTGAAGGACTACTTCCCCGAACCGGTC

ACCGTGTCCTGGAACAGCGGAGCCCTGACCTCGGGAGTCCACACTTTCCCCGCTGTG

CTGCAGTCGTCCGGCCTGTACTCGCTCTCGTCCGTGGTCACTGTCCCGTCCTCGTCC C

TGGGTACTCAGACCTACATTTGCAACGTCAACCACAAGCCTTCAAACACGAAAGTGG

ACAAGAAGGTCGAGCCGAAGTCCTGCGACAAAACCCATACTTGCCCTCCTTGTCCGG

CTCCCGAACTGCTGGGCGGACCTTCCGTGTTCCTCTTCCCGCCTAAGCCGAAAGACA C

CCTGATGATCAGCAGGACTCCGGAAGTGACATGCGTGGTGGTGGACGTGTCGCACGA

GGACCCGGAGGTCAAGTTTAATTGGTACGTGGACGGAGTGGAAGTCCACAACGCCA

AGACCAAGCCACGGGAAGAACAGTACAATTCCACCTATCGCGTGGTGTCCGTGCTTA

CCGTGCTTCACCAAGACTGGCTGAACGGAAAGGAGTACAAGTGCAAAGTGTCAAAC

AAAGCCCTGCCTGCCCCAATCGAAAAGACCATCAGCAAGGCCAAGGGGCAGCCTCG

GGAACCCCAAGTGTACACTCTCCCGCCGTCAAGAGATGAACTGACCAAGAACCAAGT

GTCCCTCACTTGTCTCGTGAAGGGATTCTACCCCTCCGATATCGCCGTGGAGTGGGA A

TCCAACGGGCAACCCGAGAACAACTACAAGACCACCCCTCCGGTGCTTGATTCCGAT

GGCTCCTTCTTCCTCTACTCCAAGCTGACCGTGGACAAGTCAAGATGGCAGCAGGGG

AACGTGTTCTCCTGCTCCGTCATGCACGAGGCCCTGCACAACCATTACACCCAGAAG

TCTCTGTCGCTGAGCCCGGGAAAATAATGAGGATCCCCCTATTCTATAGTGTCACCT A

AATGCTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTT G

TTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTT CC

TAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGG

GGTGGGGTGGGGC A GG A C A GC A A GGGGG A GG A TTGGG A A G A C A A T A GC A GGC A TG

CTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGA

GCGGCCGCAGATTGTACCTTCTGAGGCGGAAAGAACCAGCTGTGGAATGTGTGTCAG

TTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCAT

CTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGT Table 3B; Exemplary Melanin Antibody Expressing Plasmid Nucleotide Sequences

ATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCC

ATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATT T

TTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGT G

AGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTACCATGATTGAACAAGA

TGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTG

GGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGG

GCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGA

CGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCT

CGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGC

AGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATG C

AATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAA

ACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGA

TCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGC

GCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAA

TATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGT G

GCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGC

GGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAG C

GCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGGGATCGCGGAGATCCAGA C

ATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAA

ATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTG CA

ATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGG

TGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATG

AGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGG T

CATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAG C

CGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAAT

TGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTA A

TGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCC

TCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCAC T

CAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATG

TGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTT

TTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAG

GTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCT

CGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCC TT

CGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGG T

CGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGC

CTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACT G

GCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGA

GTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTG

CGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAA

ACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAG

AAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTG

GAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCAC Table 3B; Exemplary Melanin Antibody Expressing Plasmid Nucleotide Sequences

CTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAA

ACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGT C

TATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGG A

GGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGC

TCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTC

CTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAA G

TAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGT G

TCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGA G

TTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCG T

TGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAA

TTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAAC C

AAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATA

CGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGT

TCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAA

CCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGG T

GAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAA

ATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTA TT

GTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTC

CGCGCACATTTCCCCGAAAAGTGCCACCTGGGAAATTGTAAACGTTAATATTTTGTT A

AAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATC G

GCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAG

TTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAA

ACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTG G

GGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGA

GCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAG

GAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACA

CCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGC

AACTGTTGGGAAGGGCGAT

[0107] In some embodiments, the nucleotide sequence set forth in SEQ ID NO: 18 is utilized to produce a heavy chain of a melanin antibody.

[0108] In some embodiments, the nucleotide sequence set forth in SEQ ID NO: 19 is utilized to produce a light chain of a melanin antibody.

[0109] In some embodiments, the nucleotide sequence set forth in SEQ ID NO: 20 is utilized to produce a light chain of a melanin humanized antibody.

[0110] In some embodiments, the nucleotide sequence set forth in SEQ ID NO: 21 is utilized to produce a light chain of a melanin humanized antibody. [0111] In some embodiments, the nucleotide sequence set forth in SEQ ID NO: 22 is utilized to produce a light chain of a melanin humanized antibody.

[0112] In some embodiments, the nucleotide sequence set forth in SEQ ID NO: 23 is utilized to produce a heavy chain of a melanin humanized antibody.

[0113] In some embodiments, the nucleotide sequence set forth in SEQ ID NO: 24 is utilized to produce a heavy chain of a melanin humanized antibody

[0114] In some embodiments, the plasmid nucleotide sequence set forth in SEQ ID NO: 25 is utilized to produce a light chain of a melanin humanized antibody.

[0115] In some embodiments, the plasmid nucleotide sequence set forth in SEQ ID NO: 26 is utilized to produce a light chain of a melanin humanized antibody.

[0116] In some embodiments, the plasmid nucleotide sequence set forth in SEQ ID NO: 27 is utilized to produce a light chain of a melanin humanized antibody.

[0117] In some embodiments, the plasmid nucleotide sequence set forth in SEQ ID NO: 28 is utilized to produce a heavy chain of a melanin humanized antibody.

[0118] In some embodiments, the plasmid nucleotide sequence set forth in SEQ ID NO: 29 is utilized to produce a heavy chain of a melanin humanized antibody.

Therapeutic Uses

[0119] Provided herein are melanin antibodies for therapeutic use, for the treatment of melanoma.

[0120] Also provided herein are methods of treating melanoma comprising administering to a subject in need thereof a therapeutically effective amount of a therapeutic melanin antibody. In some embodiments, the melanoma is a primary melanoma. In some embodiments, the melanoma is a metastatic melanoma.

[0121] As used herein, a subject refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport, or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, and the like. Subjects may be male or female.

[0122] Without being bound to any particular theory, in melanoma tumors and metastases, the cellular turnover is rapid, resulting in an increase in leaky melanoma cells where melanin is accessible to the melanin antibodies. [0123] The administration of any of the therapeutic melanin antibodies provided herein may be administered in combination with other known drugs/treatments (e.g. small molecule drugs, or biologies). In some embodiments, the melanin antibodies may be administered with immune checkpoint inhibitors; in some embodiments, the immune checkpoint inhibitors are antibody-based immune checkpoint inhibitors. In some embodiments, the melanin antibodies may be administered with MEK inhibitors. In some embodiments, the melanin antibodies may be administered with Braf inhibitors. In some embodiments, the melanin antibodies may be administered with

chemotherapeutic agents. In some embodiments, the melanin antibodies may be administered with biologies-based therapies targeting cancer cell signaling pathways. In some embodiments, the melanin antibodies may be administered with microbiome modulation therapies, metabolic or nutritional therapies. The administration may be sequential or concurrent.

[0124] In some embodiments, for treatment for metastatic melanoma, the melanin antibodies may be administered in combination with immunotherapy (e.g. immune checkpoint inhibitors such as CTLA4, PDl, PDL-1 inhibitors). In some embodiments, the melanin antibody is conjugated to an agent. In some embodiments, the melanin antibody is conjugated to a radionuclide.

[0125] In vivo administration of the therapeutic melanin antibodies described herein may be carried out intravenously, intratumorally, intracranially, intralesionally (e.g. intralesional injection, direct contact diffusion), intracavitary (intraperitoneal, intrapleural, intrauterine, intrarectal), intraperitoneally, intramuscularly, subcutaneously, topically, orally, transdermally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In an exemplary embodiment, the route of administration is by intravenous injection.

[0126] A therapeutically effective amount of the therapeutic antibody will be administered. The appropriate dosage of the therapeutic antibody may be determined based on the severity of the melanoma, the clinical condition of the subject, the subject's clinical history and response to the treatment, and the discretion of the attending physician

[0127] The dosage amounts of the melanin antibodies provided herein may vary from about 1 ng/kg up to about 1000 mg/kg of a subject's body weight or more per day, depending upon the route of administration. For repeated administrations over several days or longer, depending on the severity melanoma, the treatment may be sustained until a desired suppression of symptoms is achieved. Dosage regimens may be useful, depending on the pattern of pharmacokinetic decay that the physician wishes to achieve. For example, dosing an individual from one to twenty-one times a week is provided herein. In certain embodiments, dosing frequency is three times per day, twice per day, once per day, once every other day, once weekly, once every two weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, or once monthly, once every two months, once every three months, or longer. Progress of the therapy is may be monitored by conventional techniques and assays. The dosing regimen may vary over time independently of the dose used.

Pharmaceutical Compositions

[0128] The present disclosure provides compositions comprising therapeutic melanin antibodies, In some embodiments the composition is sterile. The pharmaceutical compositions generally comprise an effective amount of the therapeutic antibody in a pharmaceutically acceptable excipient.

Diagnostic Uses

[0129] The melanin antibodies provided herein may be used for diagnostic and imaging purposes. Depending on the application, the melanin antibody may be detected and quantified in vivo or in vitro.

[0130] The melanin antibodies may be used for diagnostic purposes, either by detecting, localizing, or quantitating melanoma tumor cells, or melanin deposits in normal tissue.

[0131] The melanin antibodies provided herein are amendable for use in a variety of

immunoassays. These immunoassays include, but are not limited to enzyme-linked immunosorbent assay (ELISA), Western blot, radioimmunoassay (RIA), flow cytometry, a radioimmunoassay, an immunofluorescence assay, spectrophotometry, radiography, electrophoresis , high performance liquid chromatography (HPLC), or thin layer chromatography (TLC).

[0132] The melanin antibodies provided herein may be comprise a detectable label, for example detectable by spectroscopic, photochemical, biochemical, immunochemical, fluorescent, electrical, optical or chemical methods. Useful labels in the present invention include, but are not limited to fluorescent dyes, radiolabels, enzymes, colorimetric lables, avidin or biotin.

[0133] In some embodiments, the melanin antibody is radiolabeled with an isotope, useful for imaging by nuclear medicine equipment (SPECT, PET, or scintigraphy).

[0134] The diagnostic melanin antibodies may be used for the diagnosis of the primary melanoma, to monitor metastases, or to determine response to a treatment.

Kits and Articles of Manufacture

[0135] The present application provides kits comprising a melanin antibody, e.g. for either therapeutic or diagnostic use. In some embodiments, the kits further contain a component selected from any of secondary antibodies, reagents for immunohistochemistry analysis, pharmaceutically acceptable excipient and instruction manual and any combination thereof. In some embodiments, the kit comprises any one or more of the therapeutic compositions described herein, with one or more pharmaceutically acceptable excipient.

[0136] The present application also provides articles of manufacture comprising any one of the therapeutic or diagnostic compositions or kits described herein. Examples of an article of manufacture include vials (e.g. sealed vials).

ILLUSTRATIVE EMBODIMENTS

[0137] The invention may be defined by reference to the following illustrative enumerated embodiments.

[0138] Embodiment 1. A monoclonal antibody that specifically binds to melanin, wherein the antibody is chimeric or humanized.

[0139] Embodiment 2. The antibody of embodiment 1, wherein the antibody is chimeric.

[0140] Embodiment 3. The antibody of clam 2, wherein the antibody is a chimeric mouse- human antibody.

[0141] Embodiment 4. The antibody of embodiment 3, wherein the chimeric antibody comprises mouse variable regions and human constant regions. [0142] Embodiment 5. The antibody of any one of embodiments 1 to 4, wherein the melanin antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1.

[0143] Embodiment The antibody of any one of embodiments 1 to 5, wherein the melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 2.

[0144] Embodiment 7. The antibody of any one of embodiments 1 to 4, wherein the melanin antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2.

[0145] Embodiment 8. The antibody of embodiment 1, wherein the antibody is humanized.

[0146] Embodiment 9. The antibody of embodiment 8, wherein the antibody is a humanized form of the sequence of a mouse monoclonal antibody.

[0147] Embodiment 10. The antibody of embodiment 9, wherein the antibody is a humanized form of a mouse 8C3 antibody.

[0148] Embodiment 11. The antibody of any one of embodiments 1, and 8 to 10, wherein the melanin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

[0149] Embodiment 12. The antibody of any one of embodiments 1, and 8 to 10, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

[0150] Embodiment 13. The antibody of any one of embodiments 11 and 12, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 5.

[0151] Embodiment 14. The antibody of any one of embodiments 11 and 12, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 6.

[0152] Embodiment 15. The antibody of any one of embodiments 11 and 12, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 7. [0153] Embodiment 16. The antibody of any one of embodiments 11 and 12, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 5.

[0154] Embodiment 17. The antibody of any one of embodiments 11 and 12, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 6.

[0155] Embodiment 18. The antibody of any one of embodiments 11 and 12, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 7.

[0156] Embodiment 19. The antibody of any one of embodiments 1 to 10, wherein the heavy chain of the melanin antibody comprises at least one of the CDR sequences of SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0157] Embodiment 20. The antibody of any one of embodiments 1 to 10, wherein the light chain of the melanin antibody comprises at least one of the CDR sequences of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0158] Embodiment 21. The antibody of any one of embodiments 1 to 10, wherein the heavy chain of the melanin antibody comprises at least one of the CDR sequences of SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, and wherein the light chain of the melanin antibody comprises at least one of the CDR sequences of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

[0159] Embodiment 22. The antibody of any one of embodiments 1 to 10, wherein the heavy chain of the melanin antibody comprises the CDR sequences from SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, and/or wherein the light chain comprises the CDR sequences from SEQ ID NO: 3 or SEQ ID NO: 4.

[0160] Embodiment 23. The antibody of embodiments 1 or 8 to 10, wherein the antibody is an antigen binding fragment.

[0161] Embodiment 24. The antibody of any one of embodiments 1 to 23, wherein the antibody is a bispecific antibody. [0162] Embodiment 25. The antibody of embodiment 24, wherein the bispecific antibody comprises a first arm that targets melanin and a second arm that targets an antigen comprising an immune checkpoint inhibitor.

[0163] Embodiment 26. The antibody of embodiment 25, wherein the immune checkpoint inhibitor is CTLA4, PD-1, or PD-L1.

[0164] Embodiment 27. The antibody of any one of embodiments 1 to 26, wherein the antibody is conjugated to an agent.

[0165] Embodiment 28. The antibody of embodiment 27, wherein the agent is a radionuclide.

[0166] Embodiment 29. The antibody of embodiment 28, wherein the radionuclide is 213-Bi.

[0167] Embodiment The antibody of embodiment 28, wherein the radionuclide is 177-Lu.

[0168] Embodiment 31. The antibody of any one of embodiments 27 to 30, wherein the agent is conjugated to the antibody through a linker.

[0169] Embodiment 32. A pharmaceutical composition comprising the antibody of any one of embodiments 1 to 31 and a pharmacologically acceptable carrier.

[0170] Embodiment 33. A method for treating melanoma in a subject, comprising

administering a therapeutically effective amount of the antibody or composition of any one of embodiments 1 to 32 to a subject in need thereof; or stated in an alternative: a therapeutically effective amount of the antibody of any one of embodiments 1 to 31 or composition of embodiment 32 for use in treating melanoma.

[0171] Embodiment 34. The method of embodiment 33, or antibody or composition for use according to embodiment 33 wherein the melanoma is metastasized.

[0172] Embodiment 35. The method of embodiment 33, or antibody or composition for use according to embodiment 33 or 34 wherein the administration selectively induces the cell death of melanoma cells.

[0173] Embodiment 36. The method of embodiment of any one of embodiments 33, 34 or 35, or antibody or composition for use according to any one of embodiments 33 to 35 comprising administering to the subject an effective amount of at least one additional agent.

[0174] Embodiment 37. The method of, or antibody or composition for use according to embodiment 36, wherein the agent is an immune checkpoint inhibitor. [0175] Embodiment 38. The method of, or antibody or composition for use according to embodiment 37, wherein the immune checkpoint inhibitor is selected from CTLA-4, PD-1, and PDL-1.

[0176] Embodiment 39. The method of, or antibody or composition for use according to any one of embodiments 33 to 38, wherein the antibody or composition is administered intravenously.

[0177] Embodiment 40. A method of making a conjugated antibody comprising conjugating the antibody any one of embodiments 1 to 31 to an agent.

[0178] Embodiment 41. The method of embodiment 40, wherein the agent is a radionuclide.

[0179] Embodiment 42. The method of embodiment 41, wherein the radionuclide is 213-Bi.

[0180] Embodiment 43. The method of embodiment 41, wherein the radionuclide is 177-Lu.

[0181] Embodiment A polynucleotide encoding the amino acid sequence of an antibody of any one of embodiments 1 to 31.

[0182] Embodiment 45. The polynucleotide of embodiment 44, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 17.

[0183] Embodiment 46. The polynucleotide of embodiment 44, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 18.

[0184] Embodiment 47. The polynucleotide of embodiments 44 to 46, wherein the sequence has been codon optimized for expression in a human.

[0185] Embodiment 48. A vector comprising the polynucleotide of embodiment 44.

[0186] Embodiment 49. A cell line comprising the vector of embodiment 48.

[0187] Embodiment 50. A clonal cell expressing any one of the antibodies of embodiments 1 to 31.

[0188] Embodiment 51. A kit comprising any one of the antibodies or compositions of embodiments 1 to 32.

[0189] The following examples are included for illustrative purposes and are not intend to limit the scope of the invention.

EXAMPLES

Example 1; Construction and in vitro testing of chimeric and humanized melanin antibodies [0190] A mouse-human chimeric antibody was generated from the 8C3 murine monoclonal IgG melanin antibody (NCBI GenBank accession number KX346264; Uran ME, Nosanchuk JD, Restrepo A, Hamilton AJ, Gomez BL, Cano LE. Detection of antibodies against Paracoccidioides brasiliensis melanin in in vitro and in vivo studies during infection. Clin Vaccine Immunol. 2011 Oct; 18(10): 1680-8). The chimeric antibody has human constant regions, and mouse variable regions. The chimeric 8C3 antibody is interchangeably referred to herein as "8C3 Chimera" or "Chimeric 8C3" or "Chimeric 8C3 hlgGl").

[0191] Two recombinant expression vectors encoding heavy and light chains of the 8C3-hIgGl chimeric antibody were produced (pABl l-8C3-hIgGl and pAB2-8C3-hKappa, FIG. 4). These vectors were then transfected into mammalian host cells using standard techniques.

[0192] Recombinant expression vectors encoding two gamma heavy chains and three kappa light chains of the humanized 8C3 antibody were produced. (FIG. 4)

[0193] Upon expressing the heavy and light chain portions of the antibody, the mammalian host cells secreted the resulting proteins into the host medium. The antibodies were then recovered from the host cell medium in which the host cells were cultured using standard techniques.

[0194] A collection of humanized 8C3 heavy and light chains were generated.

[0195] In vitro activity of the chimeric and humanized antibodies were assessed by an ELISA assay. Sepia officinalis-derived melanin (Sigma St. Louis, MO, Sigma Cat#M2649-100MG, Lot#103H 1023V, 5mg/mL in PBS). Eight, five- fold, serial dilutions were performed on each test sample, beginning at 80 ug/mL. (10 ug melanin/well A single assay plate was used to test all six humanized antibodies, the mouse 8C3 parent antibody, the chimeric 8C3 antibody, and the mouse and human IgGl negative control antibodies. Biotinylated Goat Anti-human IgG Fc and Goat Anti- mouse-Fc antibodies were used. Streptavidin-HRP was used to detect both mouse and humanized biotinylated antibodies, and was also used to detect biotinylated chimeric 8C3. The Streptavidin- HRP (Thermo Fisher Scientific, Waltham, MA) was diluted 1 : 1000 from lmg/mL to detect the binding of biotinylated chimeric 8C3 to melanin. Biotinylated goat anti-mouse IgG-Fc (ABCAM, Cambridge, UK) or biotinylated goat anti-human IgG-Fc (ABCAM, Cambridge, UK) were diluted 1 : 1000 from lmg/mL to bind the mouse control or the human 8C3 and human controls,

respectively, and the streptavidin-HRP was used for detection. The optical density (OD) of the well contents was read on a fluorescent plate reader using 450nm emission filters. A curve-fit program was used to generate a standard curve, from which sample and control concentrations were interpolated.

[0196] Table 4 shows the test samples. (HE refers to humanized antibodies).

Table 4

[0197] FIGS. 1 and 2 show the results of the binding of the chimeric 8C3 and humanized 8C3 antibodies to melanin, as assayed in separate experiments. In these assays, chimeric 8C3 demonstrates stronger binding to melanin from Sepia officinalis than the humanized 8C variants (8C3 HE-1 through 8C3 HE-6).

[0198] Table 5 shows the tabulated results of the average absorbance values at antibody concentrations of lC^g/mL. These results correspond to the assay presented in FIG. 1.

Table 5

[0199] Table 6 shows the tabulated results of the average absorbance values at antibody concentrations of 16μg/mL. These results correspond to the assay presented in FIG. 2. Table 6

[0200] FIGS. 2 and 3 show the binding of chimeric 8C3 and parent mouse 8C3 antibodies to melanin from Sepia officinalis. FIG. 3 demonstrates stronger binding to melanin than mouse 8C3, and the average absorbance values for the test samples is provided in Table 7.

Table 7

Chimeric 8C3-hIgGl (ng/ml)*

[0201] FIG. 3 is a graph showing dose-dependent binding of mouse 8C3 to melanin.

[0202] FIG. 4 provides schematic diagrams of the plasmids used for expression of the heavy and light chains of the chimeric and humanized antibodies.

[0203] FIG. 5 shows the alignment of the chimeric 8C3 heavy chain's amino acid sequence (8C3- hlgGl chimera) and predicted complementarity-determining regions (CDR; shown in bold) with those of the two humanized 8C3 heavy chains (VH3A and VH3B). FIG. 6 shows the alignment of the chimeric 8C3 light chain's (8C3-hKappa Chimera) amino acid sequence and predicted complementarity-determining regions (CDR; shown in bold) with those of the three humanized 8C3 light chains (VK1 A, VK1B, VK4). The consensus sequences for the heavy and light chains, respectively, are listed below the sequence alignments.

[0204] Table 8 provides chemical and physical properties of the humanized antibodies, using the ExPasy ProtParam tool.

Table 8: Chemical and Physical Properties of the Humanized Antibodies

8C3-HE-(VH3A-VK4VhIgGl

Number of amino acids: 1342

Molecular weight: 147311.11

Theoretical pi: 7.3

Extinction coefficient:

Extinction coefficients are in units of M ^"1 cm ^"1 , at 280 nm measured in water.

Ext. coefficient 218360 Abs 0.1% (=1 g/1) 1.482. assuming all pairs of Cys residues form cystines

8C3-HE-(VH3A-VKlAVhIgGl

Number of amino acids: 1342

Molecular weight: 147301.16

Theoretical pi: 7.91

Extinction coefficient:

Extinction coefficients are in units of M ^"1 cm ^"1 , at 280 nm measured in water.

Ext. coefficient 218360 Abs 0.1% (=1 g/1) 1.482. assuming all pairs of Cys residues form cystines

8C3-HE-fVH3A-VKlBVhIgGl

Number of amino acids: 1342

Molecular weight: 147271.13

Theoretical pi: 8.09

Extinction coefficient:

Extinction coefficients are in units of M ^"1 cm ^"1 , at 280 nm measured in water.

Ext. coefficient 218360 Abs 0.1% (=1 g/1) 1.483. assuming all pairs of Cys residues form cystines

8C3-HE-fVH3B-VK4VhIgGl Number of amino acids: 1342

Molecular weight: 147311.11

Theoretical pi: 7.32

Extinction coefficient:

Extinction coefficients are in units of M ^"1 cm ^"1 , at 280 nm measured in water.

Ext. coefficient 218360 Abs 0.1% (=1 g/1) 1.482. assuming all pairs of Cys residues form cystines

8C3-HE-(VH3B-VKlA)-hIgGl

Number of amino acids: 1342

Molecular weight: 147321.24

Theoretical pi: 8.09

Extinction coefficient:

Extinction coefficients are in units of M ^"1 cm ^"1 , at 280 nm measured in water.

Ext. coefficient 218360 Abs 0.1% (=1 g/1) 1.482. assuming all pairs of Cys residues form cystines

8C3-HE-(VH3B-VKlB)-hIgGl

Number of amino acids: 1342

Molecular weight: 147291.22

Theoretical pi: 8.24

Extinction coefficient:

Extinction coefficients are in units of M ^"1 cm ^"1 , at 280 nm measured in water.

Ext. coefficient 218360 Abs 0.1% (=1 g/1) 1.483. assuming all pairs of Cys residues form cystines

Example 2; In Vivo Testing; Determination of antibody tissue biodistribution

[0205] For radiolabeling with ^U1 lndium, the anti-melanin antibodies (humanized 8C3 (HE-5, see Table 6, mouse 8C3, and chimeric 8C3) and control IgGl antibody were first conjugated to the bi- functional chelating agent CHXA" {N-[2-amino-3-(p-isothiocyanatophenyl)propyl]-trans- cyclohexane-l,2-diamine-N,N',N",N"',N""-pentaacetic acid} using standard methods. The CHXA" ligand was used in a 2-fold molar excess with respect to the antibodies. The antibodies were next radiolabeled with ¹¹ indium according to standard methods. The ¹¹ indium had a specific activity of 2μα^.

[0206] One million Bl 6-F10 murine melanoma cells were suspended in tissue culture medium containing Matrigel according to standard protocol. The cells were injected into the right flank of C57BL/6 mice per standard procedure. On day four (post-injection), palpable tumors were observed.

[0207] Tissue biodistribution of radiolabeled humanized 8C3 HE-5, mouse 8C3, and chimeric 8C3 antibodies was measured in various organs eight days post-tumor cell engraftment. The uptake was calculated in terms of injected dose per gram tissue (ID/g, %) according to standard procedure. The uptake of the radiolabeled antibodies was measured at two different time points following intravenous injection of the aforementioned antibodies: four hours and twenty-four hours.

[0208] The amount of radiolabeled humanized 8C3 HE-5, mouse 8C3, and chimeric 8C3 antibodies and control human IgGl antibody that bound the tumor was calculated in terms of a tumor-to-blood ratio per standard methods. Each tumor-bearing mouse received 30μΟ of ⁱⁿ Indium-mAb, and the amount of circulating (i.e. non-tumor bound) radiolabeled antibody post- injection was determined at two different time intervals: four hours and twenty-four hours.

[0209] FIG. 7 shows a representative C57BL/6 mouse bearing a B16-F10 melanoma tumor (indicated by the black circle) prior to undergoing any mAB-based anti-melanin or control treatment. FIGS. 8A-8D depict the results of a biodistribution experiment that compared the uptake of radiolabeled melanin-binding antibodies in various organs to that of a non-specific human IgG antibody control at two different time points post-antibody injection (4 hours and 24 hours). The uptake was calculated in terms of injected dose per gram tissue (ID/g, %). Compared to the tumor uptake of the chimeric 8C3 and the humanized 8C3 anti-melanin antibodies (which were both similar), the tumor uptake of the mouse 8C3 antibody was higher. In melanin-containing organs (such as the eyes and tail), the uptake of the mouse, humanized and chimeric 8C3 melanin antibodies was similar to that of the human IgG antibody control.

[0210] FIG. 9 shows the results of a tumor-to-blood ratio calculation, which provides a proxy measurement of the amount of radiolabeled melanin-binding antibodies that have bound the tumor. Although the tumor-to-blood ratio of the murine 8C3 antibody was higher than that of the humanized and chimeric 8C3 antibodies at the four-hour time point, the murine, humanized and chimeric 8C3 antibodies demonstrated similar tumor-to-blood ratios at the twenty-four-hour time point.

Example 3; Detailed Biodistribution of humanized 8C3 HE-5 for subsequent mouse and human dosimetry calculations

[0211] All animal studies were approved by the Animal Research Ethics Board of the University of Saskatchewan. For the imaging study 6 weeks old C57BL6 female mice obtained from Charles River Laboratories (USA) were injected subcutaneously with 5 x 10 ⁵ B16-F10 murine melanoma cells in Matrigel (Corning, USA) into the right flank.

[0212] Conjugation of BCA CHXA" to 8C3 HE-5. 1 OX conjugation buffer (0.05 M

Carbonate/Bicarbonate, 0.15 M NaCl, 5 mM EDTA, pH 8.6 - 8.7), 5 mL is combined with 0.5 M EDTA, pH = 8.0 (0.5 mL) and was diluted to 50 mL in a 50 mL Falcon tube with deionized water to give the IX buffer. An Ami con Ultra 0.5mL centrifugal filter (3 OK MW cut off, Fisher) was loaded with 2 mg of the humanized 8C3 HE-5 (h8C3 HE-5) antibody. The antibody was exchanged into the above conjugation buffer by performing 6 x 1.5 mL washes using an Amicon concentrator in a refrigerated centrifuge at 4°C. The final volume should be around 250 μΕ containing 2 mg of the antibody. As the buffer exchange was getting close to completion, a solution of bifunctional CHXA" ligand with 2 mg/mL concentration is prepared by dissolving CHXA" in conjugation buffer. The antibody was recovered from the Amicon and 23.6 μΕ of 2 mg/mL CHXA" solution in conjugation buffer is added to provide 5 fold molar excess of CHXA" over the antibody. The reaction mixture was incubated at 37°C for 1.5 hrs. The reaction mixtures is then purified into 0.15 M ammonium acetate buffer, pH=6.5-7.0, with 6 x 1.5 mL washes on Amicon concentrators in a refrigerated centrifuge at 4°C. The sample are stored at 4°C. A Bradford assay was performed to determine protein recovery and concentration.

[0213] Radiolabeling of antibody-CHXA" conjugate with ^m Indium ( ^m In). The radiolabeling of an antibody-CHXA" conjugate ^U1 ln was performed to achieve the specific activity of approximately 5 μCi/μg of the antibody. 600 μθ of ^U1 ln chloride was added to 10 μΕ 0.15 M ammonium acetate buffer and added to a microcentrifuge tube containing 120 μg of the h8C3 HE-5- CHXA" conjugate in 0.15 M ammonium acetate buffer. The reaction mixture was incubated for 60 min at 37°C, and then the reaction was quenched by the addition of 3 μΐ. of 0.05 M EDTA solution. The percentage of radiolabeling was measured by SG-iTLC using 0.15 M ammonium acetate buffer as the eluent (top containing unlabeled ^m In, bottom containing protein conjugated ^m In). SG-iTLCs were read on a Perkin Elmer 2470 Automatic Gamma Counter.

[0214] The biodistribution. When the tumors in mice reached approximately 200 mm ³ , the mice were randomized into the groups of 5 animals and injected IV via the tail vein with 50 of

11 lln- h8C3 HE-5. At the pre-determined time points of 1, 2, 24, 48 and 72 hrs post-injection of the radiolabeled antibody the mice were humanely sacrificed, their major organs, blood, and tumors removed, weighted, and counted in Perkin Elmer 2470 Automatic Gamma Counter (see FIG.

10). The results of the biodistribution were used for mouse and human dosimetry calculations for the proposed therapeutic radionuclides 213Bi and 177Lu.

Example 4; Human dosimetry calculations for 213Bi- and 177Lu-labeled h8C3 HE-5

[0215] This follow-up example presents dosimetry results for Bi-213 and Lu-177 in the human, extrapolated hypothetically from mouse data. The method described below is a method for extrapolating radiation dose results from mouse to human.

Methods

[0216] The extrapolation was performed by recalculating the residence times for the human model from the mouse model, and calculating the human doses using a MIRD schema

implementing software such as OLINDA1.1. The method assumes proportionality based on weig differences between species (Kirschner AS, Ice RD, Beierwaltes WH, "Radiation-dosimetry of I- 131-19-iodocholesterol: J Nucl Med. 16:248-249; 1975),

where ¾ is the recalcula ed human residence time for an ot%m or tissue, ¾j h the originally

calculated mouse residence time, the uman organ weight, i¾ the mouse organ weight, ½

is the human body weight, and ¾ is the mouse body weight. [0217] Using OLINDA ver. 1.1, the organ or tissue absorbed doses for Bi-213 were calculated and for Lu-177 using the recalculated human residence times obtained from the method stated above. For bismuth-213, which has a branching decay chain, contributions from daughter products Po-213 (97.9%) and Tl-209 (2.1%) with doses from Bi-213 were summed. In this calculation, the absorbed dose to normal organs and tissues in centigray per millicurie administered (cGy/mCi) does not include any multiplier for quality factor or relative biological effectiveness for the alpha emissions from Bi-213 and Po-213.

[0218] The tumor is not a target organ in the output results from OLINDA1.1, but it may be calculated separately using the same method as for the normal organs and tissues. For calculating tumor dose in units of centigray-equivalent per unit mCi administered, all of the absorbed doses attributed to alpha emissions were multiplied by an arbitrary factor of 5 (see for example, Sgouros et al, 1999 [Reference: Sgouros G, Ballangrud AM, Jurcic JG, McDevitt MR, Humm JL, Erdi YE, Mehta BM, Finn RD, Larson SM, Scheinberg DA, "Pharmacokinetics and dosimetry of an alpha- particle emitter labeled antibody: 213Bi-HuM195 (anti-CD33) in patients with leukemia," J Nucl Med. 40(l l): 1935-46; 1999] and Jurcic et al, 2002 [Reference: Jurcic JG, Larson SM, Sgouros G, McDevitt MR, Finn RD, Divgi CR, Ballangrud AM, Hamacher KA, Ma D, Humm JL, Brechbiel MW, Molinet R, and Scheinberg DA, "Targeted a-particle immunotherapy for myeloid leukemia," Blood 100: 1233-1239; 2002]). No such multiplier is needed for calculating the absorbed dose to tumor tissue from lutetium-177, which lacks alpha particles. To obtain the absorbed dose to tumor tissue for Bi-213 in conventional units, one may divide the centigray-equivalent dose by a factor of five to yield cGy/mCi administered to obtain the absorbed dose in cGy/mCi.

[0219] An additional caveat concerns the dose to human stomach, small intestines, and large intestines. In the MIRD schema, these organ doses are calculated using only the residence times (that is, the time-integrated activity coefficient values) obtained from radioactivity in the cavity contents, not from the cavity tissues. The mouse data represented activity in stomach and intestinal tissues (not temporary contents), and therefore it was assumed that the stomach, small intestines, and large intestines were part of the "remainder" tissues. The remainder includes all tissues in the mouse for which there was not a specific measurement for dosimetry. For example, activity in the mouse tail would be considered part of the remainder of whole body as applied by the method above to calculate the human dosimetry. The eyes are also part of the remainder, as are the other organs listed in the OLINDAl .1 output that were not specifically analyzed in the mouse study with In-111.

[0220] Blood is a transfer compartment and not a specified organ or tissue in the MIRD schema, so one does not calculate a specific dose to blood in OLINDAl .1. Dose to blood may be calculated directly in the mouse, however, but one does not extrapolate that dose to the human in OLINDAl .1.

[0221] In the following results (Table 9) the dose contributions from Bi-213 (plus daughters) and from Lu-177 are given for alpha particles, beta particles, photons, and total. All results are given to three significant figures in E-notation. The anthropomorphic model selected was the human adult. The numeric column is the equivalent of the Total column.

Table 9

Thymus 7.19E-02 1.34E-02 1.55E-03 8.68E-02 0.0868

Thyroid 7.19E-02 1.34E-02 1.55E-03 8.68E-02 0.0868

Urinary Bladder Wall 7.19E-02 1.34E-02 2.05E-03 8.73E-02 0.0873

Uterus 7.19E-02 1.34E-02 2.30E-03 8.75E-02 0.0875

Total Body 7.41E-02 1.38E-02 1.42E-03 8.93E-02 0.0893

Centigray-equivalent dose per mCi administered, a pha multiplier = 5

Tumor 2.93E-01 3.02E-03 1.32E-03 2.98E-01 0.298

Lu-177

Target Organ Beta Photon Total (Numeric)

Adrenals 2.21E-01 2.69E-02 2.48E-01 0.248

Brain 1.17E-02 1.13E-02 2.29E-02 0.023

Breasts 2.21E-01 1.65E-02 2.38E-01 0.238

Gallbladder Wall 2.21E-01 2.86E-02 2.50E-01 0.250

Lower Large

Intestine Wall 2.21E-01 3.17E-02 2.53E-01 0.253

Small Intestine 2.21E-01 3.51E-02 2.56E-01 0.256

Stomach Wall 2.21E-01 2.71E-02 2.48E-01 0.248

Upper Large

Intestine Wall 2.21E-01 3.32E-02 2.54E-01 0.254

Heart Wall 1.33E-02 2.32E-02 3.65E-02 0.037

Kidneys 6.90E-02 2.10E-02 9.00E-02 0.090

Liver 1.49E-01 2.05E-02 1.69E-01 0.169

Lungs 1.18E-02 1.84E-02 3.02E-02 0.0302

Muscle 1.58E-02 1.93E-02 3.51E-02 0.0351

Ovaries 2.21E-01 3.32E-02 2.54E-01 0.254

Pancreas 1.09E-03 2.48E-02 2.59E-02 0.0259

Red Marrow 1.64E-01 2.38E-02 1.88E-01 0.188

Osteogenic Cells 7.12E-01 4.43E-02 7.56E-01 0.756

Skin 2.21E-01 1.25E-02 2.34E-01 0.234

Spleen 7.90E-03 1.89E-02 2.68E-02 0.0268

Testes 2.21E-01 2.11E-02 2.42E-01 0.242

Thymus 2.21E-01 2.26E-02 2.44E-01 0.244

Thyroid 2.21E-01 2.30E-02 2.44E-01 0.244

Urinary Bladder Wall 2.21E-01 2.92E-02 2.50E-01 0.250

Uterus 2.21E-01 3.39E-02 2.55E-01 0.255 Total Body 2.32E-01 2.16E-02 2.53E-01 0.253

Tumor 3.14E-01 2.23E-02 3.36E-01 0.336

Example 5; Mouse dosimetry calculations for 213Bi- and 177Lu-labeled h8C3 HE-5

[0222] Using the In-111 tracer biokinetic data (decay corrected), the radiation doses from Bi-213 and Lu-177 in mice were calculated by assuming either Bi-213 or Lu-177 in place of In-111. I plotted the recalculated effective data for Bi-213 and Lu-177, obtained a best-fit mathematical function for the plotted data points, integrated the best-fit function for each source organ or tissue, and multiplied by the equilibrium dose constant and specific absorbed fraction.

[0223] The mouse data was back-decay-corrected (percent administered activity per gram tissue) to obtain the effective data (related to actual counts) for Bi-213 (half-life is 45.6 minutes) and for Lu-177 (half-life is 160 hours). For each organ or tissue, the effective data points were plotted against sampling time, and linear least-squares regression analysis was perofrmed to obtain a best- fit single (or double) exponential function to the data, with best-fit equation parameters.

[0224] Next, the exponential function was integrated to obtain an estimate of the microcurie-hours per microcurie administered, represented by the area under the time-activity function, integrated to infinity (complete decay) for both the Bi-213 and the Lu-177 cases. It was assumed that the Bi-213 absorbed fraction was 1.0 for all emissions in the mouse organs and tissues. Model values for Lu- 177 emissions were calculated for fraction of energy emitted from the measured organ or tissue that deposits in the same organ or tissue using the mouse model developed earlier by Miller et al. (Miller WH, Hartmann-Siantar C, Fisher DR, Descalle M-A, Daly T, Lehmann J, Lewis MR, Hoffman T, Smith J, Situ PD, and Volkert WA, "Evaluation of Beta Absorbed Fractions in a Mouse Model for ⁹⁰ Y, ¹⁸⁸ Re, ¹⁶⁶ Ho, ¹⁴⁹ Pm, ⁶⁴ Cu, and ¹⁷⁷ Lu Radionuclides." Cancer Biother. & Radiopharm.

20(4):436-449; 2005).

[0225] Equilibrium dose constants for Bi-213 and Lu-177 were obtained from Eckerman KF and Endo A, MIRD Radionuclide Data and Decay Schemes, 2 ^nd ed., Reston, Virginia: Society of Nuclear Medicine; 2008. For Bi-213, the equilibrium dose constant is 19.44 g cGy uCi ^"1 hr ^"1 , and for Lu-177, the equilibrium dose constant is 0.315 g cGy uCi ^"1 hr ^"1 . With the equilibrium dose constant, the absorbed fraction of emitted beta energy, and the integral activity residing in the organ or tissue through complete decay all known or calculated, the absorbed dose in units of cGy (centigray) per microcurie (cGy/uCi) administered Bi-213 and Lu-177 was then calculated to obtain the following results (average dose and correlation coefficient):

Results for mouse organs are shown in Table 10:

Table 10

[0226] The Pearson product-moment correlation coefficient (r) is a measure of the strength and direction of the linear relationship between two variables defined as the covariance of the variables divided by the product of their standard deviations, and indicates the correlation between the data and the mathematical function that was used to integrate the area-under-curve to determine the number of radioactive transitions taking place in the organ or tissue (integrated to infinity). The r values for Bi-213 are high because of its very short half-life, and which gave three time points for curve-fitting. Example 6; Comparative therapy of B16-F10 melanoma tumors with 213Bi- versus 177Lu- labeled h8C3 HE-5 antibody

[0227] 213Bi/225Ac generator was purchased from Oak Ridge National Laboratory (TN, USA), 177Lu chloride - from Radiomedix (TX, USA). The h8C3 HE-5 antibody was conjugated to CHXA" bifunctional ligand as described in Detailed Biodistribution. The antibody was radiolabeled with 213Bi which was eluted from 213Bi/225Ac generator immediately prior to the radiolabeling in form of 213Bi iodide or with 177Lu. The radiolabeling of an antibody-CHXA" conjugate with 213Bi or Lu was performed to achieve the specific activity of approximately 5 μCi/μg of the antibody. To prepare a "high" (400 μθ) dose of 213Bi- or 177Lu-labeled antibody, 400 μθ of a radionuclide solution in 0.15 M ammonium acetate buffer was added to 80 μg of the antibody- CHXA" conjugate; to prepare a "low" (200 μθ) dose of 213Bi- or 177Lu- labeled antibody, 200 μθ of a radionuclide solution in 0.15 M ammonium acetate buffer was added to 40 μg of the antibody-CHXA" conjugate. For labeling with 213Bi the reaction mixture was incubated for 5 min at 37oC, for labeling with 177Lu - for 60 min. The incubation was followed by quenching the reaction by the addition of 3 μΐ _^ of 0.05 M EDTA solution. The percentage of radiolabeling was measured by SG-iTLC using 0.15 M ammonium acetate buffer as the eluent (top containing free radionuclide, bottom containing radiolabeled antibody). SG-iTLCs were read on a Perkin Elmer 2470 Automatic Gamma Counter.

[0228] Female C57B16 mice were injected with 5x105 B16-10 melanoma cells into the right flank as described in example 3. The mice were used for therapy when their tumors reached

approximately 50 mm ³ . The mice were randomized into the group of five animals and treated with either: high dose of 213Bi-h8C3 HE-5, or low dose of 213-h8C3 HE-5, or high dose of 177Lu-h8C3 HE-5, or low dose of 177Lu-h8C3 HE-5, or 80 μg unlabeled ("cold") h8C3 HE-5, or left untreated. Their tumors were measured every three days with electronic calipers to calculate the tumor volume for 21 day (FIGS. 11 A and 1 IB). The mice were weighed every 3 days (FIG. 14A and 14B). Their blood was analyzed on a weekly basis for white blood cells (FIG. 12A and 13 A), red blood cells (FIG. 12B and 13B) and platelet count (FIG. 12C and 13C). At the completion of the experiment mice were sacrificed and their blood was analyzed for ALT (FIG. 15A), AST (FIG. 15B), urea (FIG. 15C) and creatinine (FIG. 15D). [0229] The 213Bi- and 177Lu-labeled h8C3 HE-5 antibody efficacy in radioimmunotherapy of B16-F10 melanoma were compared. The results of the experiments demonstrated that short-lived (46 min physical half-life) alpha-emitter 213Bi was much more efficient in killing melanoma cells than long-lived (6.7 days physical half-life) beta-emitter 177Lu. Without being bound to any theory, the superior efficiency of 213Bi delivered by h8C3 HE-5 to the melanoma tumors may be explained by a better match between fast dose rate of 213Bi decay and aggressive growth of B16-F10 cells (doubling time 7hrs) while slower decaying 177Lu needs a longer time to deliver its radiation dose and cannot match this cell growth. The relative biological effectiveness (RBE) of alpha-particles emitted by 213Bi is several times higher than that of beta-particles, thus resulting in more efficient tumor control.

Example 7; Fractionation therapy with 213Bi-h8C3 HE-5

[0230] The same murine melanoma model as in Comparative Treatment was used. h8C3 HE-5 antibody was radiolabeled with 213Bi as in Comparative treatment. Tumor-bearing mice were randomized into the groups of 8 and treated with either: single dose 400 μθ 213-h8C3 HE-5 on Day 0, or 400 μθΐ 213-h8C3 HE-5 on Day 0 and on Day 3, or 400 μθΐ 213-h8C3 HE-5 on Day 0, Day 3 and Day 7. On Day 16 mice in the single dose group were treated with another 400 μCi 213- h8C3 HE-5 dose. Changes in tumor volume are depicted in FIGS. 16A, 16B, and 16C. Changes in mouse body weight are depicted in FIG. 17. Comparative blood counts for white blood cells, red blood cells, and platelets are depicted in FIGS. 18A, 18B, and 18C, respectively. Systemic toxicity to the kidney and liver are depicted in FIG. 19.

Example 8; microSPECT/CT imaging of B16-F10 melanoma tumor bearing mice with lllln- h8C3 HE-5

[0231] The mouse model and radiolabeling with 11 lln of h8C3 HE-5 antibody were performed as described. microSPECT/CT (micro single photon emission computer tomography /computer tomography) images were collected on a MILabs VECTor4 (Netherlands) microSPECT/CT scanner and processed using the comprehensive image analysis software package PMOD (version 3.9, PMOD Technologies, Inc, Switzerland). Imaging studies were conducted using 200 μCi 11 lln at a 5: 1 mCi/mg specific activity with a CHXA" conjugated h8C3 HE-5. Two tumor-bearing mice were injected IV via tail vein and imaged in the prone position at 1, 24, 48, 72, and 216 hours post injection (FIG. 20). SPECT data was collected for 20 minutes using an Extra Ultra High Sensitivity Mouse (XUHS-M) collimator for 20-350 keV range using spiral trajectories. All SPECT images were reconstructed using both 245 keV and 171 keV 11 lln gamma emissions on a 0.4 mm voxel grid with MILabs reconstruction software.

Example 9; Generation of Recombinant Cell Lines Expressing 8C3 HE-5 Antibody

[0232] CHO DG44 host cells were transfected with vectors encoding h8C3 HE-5 antibody.

Transfectants were selected and subjected to one round of subcloning by limited dilution. Three subclones were selected for the generation of Research Cell Banks ("RGBs") designated as follows: SUBCLONE-2-3H2, SUBCLONE-2-20C3, and SUBCLONE-2-3H11.

Transfection and Generation of Bulk Pools and Mini-Pools

Transfection of DHFR-deficient CHO DG44

[0233] The dihydrofolate reductase (DHFR)-deficient CHO DG44 cell line used as a host for the recombinant cell lines described here is an auxotroph for hypoxanthine and thymidine (HT) that was developed by Dr. Larry Chasin of Columbia University. The DHFR- CHO line was derived from EMS and γ-radiation-induced mutations of the CHO Kl cell line ATCC CCL-61. The ATCC CCL- 61 cell line is a proline auxotroph of a cell line established from Cricetulus griseus ovarian tissue by Dr. Ted Puck in 1958. Dr. Chasin used two rounds of γ-radiation to produce a cell line completely lacking both alleles of the DHFR gene.

[0234] The DHFR- cell lines DUXB11 and DG44 have been used since 1981 for the production of recombinant proteins. More recently, the DG44 cell line has been adapted to grow in chemically defined, serum- free medium as a suspension cell line. Aragen obtained the suspension-adapted DG44 cells as a frozen culture from Invitrogen in 2008 (Gibco-Invitrogen, Cat 12609-012, lot number 288885). The cells were expanded in CHO DG44 medium (Invitrogen), a chemically defined medium, and frozen down in a mixture of that medium and 7.5% cell culture grade DMSO (Sigma). The cells were passaged in antibiotic-free medium three times and tested by NAMSA for Bacteriostasis/Fungistasis and sterility, by Research Animal Diagnostic Laboratory (RADIL) for IMPACT VII PCR profile, and by Bionique Testing Laboratories, Inc. for mycoplasma. The cells met the specified test requirements.

[0235] The plasmids, pAB2-8C3-HE-LRLC (VK1) (625.82.2 [Pvul]) and pABl 1-8C3-HE- LRMRHC (VLB) (625.85.5 [Pvul]) encoding (respectively) the antibody heavy and light chain are described herein. The plasmids also encode DHFR and neomycin selectable markers, respectively. The plasmids were linearized by overnight digestion with the restriction enzyme Pvul followed by phenol-chloroform and ethanol precipitation. Plasmid DNA was re-suspended in 0.1 x TE buffer and the concentration measured at 260nm. The DNA was adjusted to 1 μg/μL by the addition of sterile 0.1 X TE buffer.

[0236] Nine sets of Neon electroporations using 1/1 vector ratios were performed in DG44 host cells. For each transfection, a total amount of 10 μg of DNA was added to 100 μL· of CHO DG44 cells suspended in Resuspension Buffer R at a concentration of 4.0x106 cells/mL. The DN A/cell mixture was drawn into a Neon tip 100 and electroporated using the Neon electroporation device from Invitrogen with a 1700 V x 20ms x 1 pulse program. In parallel with these nine transfections, one set of transfection was performed using Aragen AB2 vector carrying the GFP sequence.

Promptly following electroporation, the transfected cells were diluted into 2 rriL of CD-DG44 medium supplemented with 8 mM Glutamax in a 6-well plate and cultured in static condition at 37°C and 5% CO2. Transfection efficiency was measured by FACS analysis of the GFP transfected cells, 72 hours after transfection. Seventy-two hours after transfection, forty six percent of the cells transfected with the GFP carrying DNA were positive for GFP by FACS analysis, which corresponded to the average transient transfection efficiency expected at that stage.

[0237] Three days after electroporation, the cells from the nine wells for each transfection were pooled and media exchanged into CD-OptiCHO (HT deficient) + 8 mM Glutamax. Next, the pools were used to generate two types of stable selected pools (bulk pools and mini-pools). Generation of Bulk Pools

[0238] Bulk pools were generated as a way to obtain CHO derived materials within a relatively short period of time (-3-4 weeks). One bulk pool was generated with gradual increase of G418 (0.25mg/mL→ 0.5 mg/mL final) and auxotrophic DHFR selection with HT deficient medium in static flasks. The bulk pool was adapted into shake flasks upon recovery of cell viability to ~ 90%.

[0239] Further, the performance of the pool was assessed in shake flasks by seeding 125 mL shake flasks at 5 xl05 cells/mL in 50 mL of CD-OptiCHO media supplemented with 8 mM

Glutamax. The shake flask was cultured at 37°C and 5% CO2, on a shaker platform equipped with a 25 mm orbital throw set up at 125 rpm. The cultures were fed with 5 % (initial culture volume) of Cell Boost 7a with lOmg/L Invitrogen recombinant human insulin and 0.5 % of Cell Boost 7b (initial culture volume) from Hyclone on Days 3 and 6 and 8. Cell number was counted (FIG. 21) and conditioned media were taken on Days 3, 6, 8 and daily after Day 9. Cultures were harvested at -80% viability by centrifuging at 2500 rpm for 5 min on day 11. The protein concentration in the conditioned media was measured by ForteBio Octet Red with a Protein A sensor using a purified IgGl antibody as a standard. The expression levels obtained from the pools are presented on FIG. 22.

[0240] Lastly, the 8C3 HE-5 antibody in the condition media was purified on Protein A drip column, the purification fractions were analyzed by SDS-PAGE.

Generation of Mini-Pools

[0241] Mini-pools were generated three days after transfection by plating the transfected pools into mini-pools at 1,000 cells per well under auxotrophic DHFR selection in CD-OptiCHO medium supplemented with 8 mM Glutamax (18 x 96-well plates) in 200 μΐ of medium, plates were cultured at 37°C and 5% CO2. Beginning three days after plating, the mini-pools were subjected to a gradual increase of G418 concentration (0.25mg/mL→ 0.5 mg/mL final) and methotrexate (MTX) (100 nM → 200 nM→ 400 nM final) through media exchange over a 4-week period. Cell confluence was monitored by microscope during this time with higher selection applied upon cell growth (i.e., increase in cell confluence). After -5 weeks, the plates were assayed by ELISA using Goat-anti- Human IgG-Fc and Goat-anti-Human kappa chain-HRP as coating and detecting antibodies, respectively (FIG. 23).

[0242] The 120-top expresser mini-pools obtained from the 96- well plate screening were expanded to 24-well plates and re-screened for expression in 24-well plates. Cells were plated in new 24-well plates at approximately 20% confluence in fresh media in CD-OptiCHO supplemented with 8 mM Glutamax. Condition media were collected on Day 7 and 11. The protein concentration in the conditioned media was measured by ForteBio Octet Red with a Protein A sensor using the 8C3 HE-5 antibody purified from the bulk pool as standard (FIG. 24).

[0243] After screening, the highest 24-well plates expresser mini-pools were pooled in three super pools. The list of mini-pools selected for the three super-pools is presented in the FIG. 25. Super- pool 1 was composed of the three highest expresser mini-pools with titers ranging from 106 to 129 μg/mL, the Super-pool 2 was composed of five mini-pools with titers ranging from 60 to 75 μg/mL and the Super-pool 3 was composed of seven mini-pools with titers ranging from 40 to 58 μg/mL.

[0244] The Super-Pools were passaged in CD-OptiCHO medium supplemented with 8 mM Glutamax, 0.5 mg/mL G418 and 400 nM MTX for approximately 2 weeks until viability approached 85%. At that time, the Super-pools were cryopreserved, processed with limited dilution and evaluated in fed batch shaker flasks for expression.

Shake Flasks evaluation of the Super Pools.

[0245] The super-pools were evaluated in fed batch shake flasks. Cells were seeded at 5x105 cells/mL in 50 rriL of CD-OptiCHO medium supplemented with 8 mM L-glutamine, in 250 ml shake flasks. The shake flasks were cultured at 37°C and 5% CO2, on a shaker platform equipped with a 25 mm orbital throw rotating at 125 rpm. The cultures were fed with 5% of Cell Boost 7a supplemented with lOmg/L Invitrogen recombinant human insulin and 0.5 % of Cell Boost 7b, daily on Day 3, 6, 8 and 10. NOVA readings were performed on Days 3, 6, 8 and as needed until harvest to monitor and adjust for glucose and L-glutamine. Cell counts, and samples of cultures were taken on Days 3, 6, 8, 10 and everyday thereafter until harvest. The cultures were harvested at < 80% viability. The growth curve and viability are presented in the FIGS. 26 and 27. Super-pool- 1 adapted slower than cells from Super-pool -2 and -3 to suspension growth in shake flasks, as a consequence two runs of fed batch evaluations were performed for Super-pool- 1. The expression profiles are presented in the FIG. 28 below. The highest expression, 792.3mg/L, was obtained with Superpool-1 repeat and super-pool 2 had 462 mg/L.

Limited Dilution of Mini-pool Derived Super-Pools

Limited Dilution and ELISA Screening Clones

[0246] Three super-pools were cloned by limited dilution method. Each culture was seeded in 96- well plates at 0.5 cells/well. Twenty 96- well plates were plated for each superpool. Cloning medium were composed of CD OptiCHO supplemented with 8mM Glutamax, 2mM Glutamine, 5μg/mL Insulin, IX HT and equal volume of condition medium collected from bulk pool culture. Plates were incubated in a static incubator at 37°C with 5% CO2 for 14 days and each well was imaged on Day 0, 1, 2, 5 or 7 and day 13 or 14 by Solentim Imaging System. Fresh medium, 1 ΟΟμΙ _^ , was added into each well on Day 7 and medium were changed on Day 14. After fourteen or fifteen days incubation, all plates were screened by ELISA using Goat-anti-Human IgG-Fc and Goat-anti- Human kappa chain-HRP as coating and detecting antibodies, respectively.

[0247] Based on Solentim images and ELISA screening results, the top 135 clones, originated from single cells were expanded up to 24-well plates in CD-OptiCHO medium supplemented with 8 mM Glutamax, 0.5 mg/mL G418 and 400 nM MTX.

[0248] The top 135 clones expanded to 24-well plates were monitored periodically with a microscope. After approximately 7 days, the wells reached 80% confluence. At this time, each clone was seeded at 20% confluence in fresh media in a well of a new 24-well plate. Cultures were incubated for 11 days in static conditions at 37°C and 5% CO2. Condition media were collected on day 7 and 11. Clones were ranked based on expression levels measured on day 11 using a ForteBio Octet Red with a Protein A sensor and compared to a standard curve obtained with the 8C3 HE-5 antibody purified from the bulk pool (FIG. 29). Based on the 24-well expression level profile, a total of 36 clones with expression levels range from 95.7 to 221.8 μg/mL were expanded into T-75 and subsequently into 125 mL shake flasks. The expression level of the top 36 clones in 24 well stage is summarized in FIG. 30. [0249] The top 36 clones expanded to shake flasks were cryopreserved (3 vials each) in 7.5% DMSO and 92.5% CD-OptiCHO media. The vials were placed into Nalgene Cryo 1°C Freezing. Container (-l°C/minute cooling rate) and stored at -80°C. After 48 hours, the vials were transferred and stored in a liquid nitrogen tank.

Shake Flask Evaluation of Top Clones

[0250] Thirty-five of the thirty-six top expressers sub-clones identified at the 24 well plates stage successfully adapted to suspension growth in shake flasks. These top sub-clones were evaluated for expression in 250 mL shake flasks in fed batch conditions. Shake flasks were seeded at 5x105 cells/mL in 50 mL of CD-OptiCHO medium supplemented with 8 mM L-glutamine. The shake flasks were cultured at 37°C and 5% CO2, on a shaker platform equipped with a 25 mm orbital throw rotating at 125 rpm. The cultures were fed with 5% of Cell Boost 7a and 0.5 % of Cell Boos 7b, daily on Day 3, 6, 8 and 10. NOVA readings were performed on Days 3, 6, 8 and daily as needed until harvest to monitor and adjust for glucose and L-glutamine. Meanwhile, cell counts, and samples of cultures were taken on Days 3, 6, 8, 10 and daily thereafter until harvest. The cultures were harvested at < 80% viability. Cells were centrifuged at 2500 rpm for 5 min and conditioned medium transferred and stored at -20°C.

[0251] Clones 2-3H2, 2-3H11, 2-11H12 and 2-20C3 reached the highest expression levels with respective expression levels of 1.29 g/L, 1.27 g/L, 1.26 g/L, and 1.25 g/L. Maximum Viable Cell Density (VCD), viability profile, titer at harvest, longevity of the cultures and clonality analyzed from Solentim images were summarized in FIG. 31.

[0252] Clones 2-3H2, 2-3H11 and 2-20C3 highlighted in FIG. 31 and were selected for the preparation of the research cell banks.

[0253] The harvest conditioned medium obtained from the five top expresser clones were analyzed by SDS-PAGE. Four microliters were loaded on each band in reduced and non-reduced condition. Expected molecular weight bands were obtained in reduced and non-reduced conditions with all five clones. Preparation of Research Cell Banks

[0254] Clones 2-3H11, 2-3H3 and 2-20C3 were selected for the preparation of Research Cell Banks (RCB), based on their expression level at harvest and clonality from Solentim.

[0255] Each clone was expanded into 250 mL and RCB was prepared by banking 36 vials with 1x107 viable cells in ImL volume of 7.5% DMSO and 92.5% CD-OptiCHO media supplemented with 8 mM GlutaMax per vial. The vials were placed into Nalgene Cryo 1 °C Freezing Container (- l°C/minute cooling rate) and stored at -80°C. All vials were transferred and stored in a liquid nitrogen tank after 48 hours.