AU2015215852A1 - Vaccine compositions - Google Patents

Abstract

The present disclosure relates to vaccine compositions, particularly KLH-peptide conjugates, and methods of making such compositions. The present disclosure 5 further relates to methods of reducing abnormal cell growth using the compositions described herein.

Description

- 1 VACCINE COMPOSITIONS The present application is a divisional application of Australian Application No. 2010334428, which is incorporated in its entirety herein by 5 reference. This application claims priority to U.S. Provisional Application No. 61/289,083 filed on December 22, 2009, which is incorporated herein by reference in its entirety. Field 10 The present disclosure relates to vaccine compositions, particularly KLH peptide conjugates, and methods of making such compositions. The present disclosure further relates to methods of reducing abnormal cell growth using the composistions described herein. Background 15 CDX-1 10 is being developed for the treatment of patients suffering from glioblastoma multiforme, a type of brain cancer. A significant portion of patients with this disease express a variant of the epidermal growth factor receptor (EGFR) on the surface of their cancer cells. This variant, known as EGFRvIII, is formed from a splice mutation leading to a unique peptide sequence (typically of 20 13 amino acids) on the surface of the cancer cells. Since this variant is not expressed on normal cells, the EGFRvIII sequence is a good target for anti cancer therapies. EGFR is a protein that has been well validated as a target for cancer therapy. Unlike EGFR, however, EGFRvIII is not present in normal tissues, 25 suggesting this target will enable the development of a tumor-specific therapy for cancer patients. Furthermore, EGFRvIII is a transforming oncogene that can directly contribute to the cancer cell growth. While originally discovered in glioblastoma multiforme (GBM), the most common and aggressive form of brain cancer, the expression of EGFRvIII has also been observed in various other 30 cancers such as breast, ovarian, metastatic prostate, colorectal, and head & neck cancers.

- 1a CDX-110 is a conjugate vaccine formed by covalently linking the EGFRvIII peptide, to which a C-terminal cysteine has been added, to the carrier protein Keyhole Limpet Hemocyanin (KLH) (see, e.g. U.S. Patent Nos. 5,401,828, 6,224,868; and WO 2007/056061). KLH also acts as an immune 5 stimulant to enhance immune responses against the EGFRvIII peptide. Chemical linkage of the EGFRvIII peptide and KLH is achieved through use of the bi-functional linker, Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1 carboxylate (Sulfo-SMCC). Previous processes for production of CDX-1 10 and formulations thereof 10 had several drawbacks for use in a commercial setting. For example, such drawbacks included lack of complete solubitilty of the linker (SMCC) resulting in a reaction that is difficult to control unacceptable levels of process impurities; process methodology that is not amenable to scale-up; lack of sufficient control of key biophysical characteristics such as the ratio of peptide:KLH carren, size heterogeneity, partiQulate and get formation, and product instability, KLH is a very large carrier protein which has 5 pre viously proved very difficult to conjugate and formulate to high pharmaceutical standards. Accordingly, there is a need for improved manufacturing methods and formulations that provide compositions of KLH-peptide conjugates that address such drawbacks. Summary 10 Other features and advantages of the instant disclosure will be apparent from the following detailed description and examples which should not be construed as limiting, The contents of all references. figures, Genbank entries, patents and published patent applications cited throughout the disclosure are expressly incorporated herein by reference in their entirety, 15 In one aspect, the disclosure provides a composition comprising a KLHpeptide conjugate, a potassum phosphate buffer, a disaccharide, and a surfactant. In one embodiment, the potassium phosphate buffer is present in a concentration ranging from 5 mM to 30 mM. for example front mM to 20 mM' from 5 MM to 15 mM from 7 mM to 13 mM, from 8 mM to 12 mM, from 9 mM to 11 mM, or from 9.5 mM to 10.5 mM. In 20 another embodiment potassium phosphate buffer is present in a concentration of 10 mM, In a further embodiment the potassium phosphate buffer is present in a concentration such that after 12 weeks at 40'G, the concentration of the KLH-peptide conjugate ir said composition, as measured in mg/mL, has changed by less than 15% when compared to the original concentration. 25 In a further embodiment, the disaccharide in the composition is trehalose. In one embodiment, trehalose is present in a concentration ranging from 45 mg/mL to 150 ng/mL, from 70 mg/n to 120 mg/mb, from 80 mg/mL to 100 mg/mL, or from 85 mg/mL to 95 mg/mL In another embodiment, trehalose is present in a concentration of 90 mg/mL, In another embodiment, trehalose is present in a concentration such that after 30 12 weeks at 40"C, the concentration of the KLH-peptide conjugate in said composition, as measured in mg/mL, has changed by less than 15% when compared to the original concentration. In a further embodiment, the surfactant in the composition is polysorbate 80. For example, in one embodiment the polysorbate 80 is present in a concentration ranging from 0,01 mgimL to 0.3 mg/mL., from 0,05 mg/mL to 0.25 mg/mL, from 0.1 mg!mL to 0.25 mg/mL, or from 0.15 mg/mL to 0.25 mg/mL. In one embodiment the polysorbate 80 is present in a concentration of 0.2 ng/mL in a further embodiment, the polysorbate 80 is present in a concentration such that after 12 weeks at 40t the concentration of 5 the KLH-peptide conjugate in said composition, as measured in rmg/mL, has changed by less than 15% when compared to the original concentration. In one aspect the present disclosure provides any of the compositions as described herein, wherein the peptide conjugated to KWH in the KLH-peptide conjugate comprises; consists essentially of, or consists of SEQ ID NO:1. in a further 10 embodiment, the peptide conjugated to KLH in the KLH-peptide conjugate comprises, consists essentially of, or consists of SEQ ID NO:2. In one embodiment, the peptide is conjugated to KLH with a sulfo-SMCC tinker. In one aspect, the present disclosure provides any of the KLH-peptide compositions described herein, wherein the epitope density ranges from 20 to 80. For 15 exarnple, in one embodiment the epitope density ranges from 25 to 75, from 25 to 70, from 25 to 65, from 30 to 60, from 30 to 55, from 35 to 50, from 40 to 50, In a further embodiment, the epitope density is 25, 26, 27 28, 29, 30, 31 32, 33, 34, 35, 36, 37, 383 39, 40, 41 42, 43, 44, 45, 46, 47, 48; 49, 50 61, 52, 53, 54, 55, 567 , 58 S9. or 60, In a further aspect, the present disclosure provides any of the compositions 20 described herein, wherein the amount of KLHpeptide conjugate present in diner form ranges from 45% to 65% by mass of the total mass of the composition, as determined by size exclusion chromatography. For example, in one embodiment the dimer form ranges from 50% to 60%, from 51% to 59%, from 52% to 58%, from 53% to 57%, or from 54% to 56% by mass, In a. further embodiment the amount of KL-peptide 25 conjugate in dimer form is 50%, 51%, 52%, 53% 54%, 55%, 56%, 57%. 58% 59% or 60% by mass of the total mass of the composition, as determined by size exdusion chromatography. In a further embodiment, the amount of KLH-peptide conjiugate in dimer form is greater than 80% by mass of the total mass of the composition, as determined by size exclusion chromatography, For example, in one embodiment, the 30 amount in dimer form is greater than 85%, greater than 90%, greater than 95%, greater than 96% greater than 97%, greater than 98%, or greater than 99% by mass, In a further aspect, the present disdosure provides any of the compositions described herein, wherein when subjected to size exclusion chromatography with detection by UV absorption, the peak corresponding to the diner forn of the KLH- -4 peptide conjugate is from 45% to 65% of the total area under the curve. For example, in one embodiment the peak corresponding to the dimer form is from 50% to 60%, from 51% to 59%, from 52% to 58%, from 53% to 57%, or from 54% to 56% of the totaarea under the curve, in a further embodiment the peak corresponding to the dier fom is 5 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60% of the total area under the curve. In a further embodiment, the peak corresponding to the diner form is greater than 80% of the total area under the curve. For example, ii ore embodiment, the peak corresponding to the dimer form is greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% of the 10 total area under the curve, In a further aspect the present disclosure provides any of the compositions descrbed herein, wherein the amount of KLH-peptide corugate present in rmonomer form ranges from 15% to 40% by mass of the total mass of the composition as determined by size exclusion chromatography. For example, in one embodiment The 15 dimer form ranges from 20% to 35%. from 21% to 34%: from 22% to 33%, from 23% to 32%, from 24% to 31% or from 25% to 30% by mass. In a further embodiment, the aront of KLH peptide conjugate in monomerform is 18%, 19%, 20%, 21%, 22%, 23%, 24 25%, 26%. 27%28%, 29%' or 30%' byvmass of the totalmass of the composition, as determined by sie exclusion chromatograp.hy. In a further embodiment, the amount 20 of KLH-peptide conjugate in monomer form is greater than 80% by mass of the total mass of the composition, as determined by size exclusion chromuatography. For example, in one embodiment, the amount in monomer form is greater than, 85% greater than 90% greater than 95%, greater than 96%, greater than 97%, greater than 98%or greater than 99% by mass. 25 In a further aspect, the present disclosure provides any of the compositions described herein, wherein when subjected to size exclusion chromatography with detection by UV absorption, the peak corresponding to the monomer form of the IK.LH peptide conjugate is from 15% to 40% of the total area under the curve. For example, in one embodiment the peak corresponding to the monomer form is from 20% to 35%, 30 front 21% to 34%, from 22% to 33%, froi 23% to 32%, from 24% to 31%, or from 25% to 30% of the total area under the curve, In a further embodiment, the peak corresponding to the monomer form is 18%, 19%, 20% 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28% 29%, or 30% of the *total area under the curve, In a further embodiment, the peak corresponding to the monomer form is greater than 80% of the total area under the curve For example in one ernbodliment, the peak corresponding to the monomer form is greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% of the total area under the curve. 5 The present disclosure further provides any of the KLH- peptide conjugate compositions described herein, wherein the composition is an aqueous pharmaceutical composition ln one embodiment, the pH of said composition ranges from 6 to 8. in a further embodiment, the. pH of the composition ranges from 6.5 to 7.5. In a further embodiment, the pH of the composition is 6,5, 6,6 6.7, .8, 7 6,9, 7., .1, 7,2, 7.3, 7.4 or 10 7.5. In a further embodiment, the pH is an amount such that after 12 weeks at 40C, the concentration of the KLH-peptide conjugate in said composition, as measured in mgimkL has changed by less than 15% when compared to the original concentration, In a further aspect, the present disclosure provides a liquid composition comprising a KLH-peptide conjugate, potassium phosphate buffer, trehalose, and 15 polysorbate 80, wherein: the peptide conjugated to KLH comprises, consists essentoaly of or consists of SEQ ID NO:1 or SEQ ID NO:2; the KLH-peptide conjugate has an epitope density ranging from 30 to 65! the buffer is present in a concentration ranging from 9 mM to 11 msI the pH of the composition ranges from 73 to 7:5; the trehadose is present in a concentration ranging from 35 mg/mL to 95 mg/mL; the polysorbate 80 is 20 present in a concentration ranging from 0. mghmL to 0.3 mg/mL; and further wherein the amount of KLH-peptide conjugate present in monomer form ranges from 18% to 35% by mass of the total mass of the composition, ard the amount of KLH-peptide conjugate present in dimer forrn ranges from 50% to 65% by mass of the total mass of the composition, as determined by size exclusion chromatography For example, in one 25 embodiment the buffer is present in a concentration of 10 mM, the pH of the composition is 7.4, the trehaLse is present in a concentration of 90 mgmL, and the polysorbate 80 is present in a concentration of 0,2 mg/mL Further, for example, said liquid composition was prepared by reconstituting a lyophilized composition with water. The present disclosure further provides a lyophilized composition comprising, 30 consisting essentially of, or consisting of a KLH-peptide conjugate, a potassium phosphate buffer, a disaccharide, and a surfactant In one embodiment, sid lyophilized composting, when reconstituted with water, has a pH ranging from 6 to 9. In one embodient, the disaccharide is trehalose and the surfactant is polysorbate 80. in a further embodiment, the trehalose is present in an amount ranging from 30 to 110 mg per mg of KI-Hpeptide consjugate; and the polysorbate 80 is present in an amount ranging from 0,01 to 0,3 mg per mg of KLH-peptide conjugate, In a further embodiment the trehalose is present in an amount of 90 mg per mg of KLH-peptide conjugate and the polysorbate 80 is present in an amount of 0,2 mg per mg of KL-Hpeptide conjugate. 5 In one aspect, the disclosure provides a liquid composition comprising a KUH EGFRvil conjguate, wherein after 12 weeks at 40C, the concentration of said conjugate in said liquid composition, as measured in mgmL, has changed by less than 15%, less than 13%, less than 11%, less than 10%, less than 8%, or less than 5% when compared to the original concentration 10 In a further aspect, the disclosure provides methods of reducing abnormal cels growth in a mammal in need thereof, comprising the step of administering to said mammal any of the compositions disclosed herein. in one embodiment, the abnormal cell growth is cancerous, In a further embodiment, the cancer is glioblastoma, In a further aspect; the disclosure provides methods of reducing abnormal cell 15 growth in a mammal in need thereof, comprising the steps of a) mixing any of the compositions disclosed herein with an ad]juvant; and b) administering to said mammal the resulting mixture. In a further aspect, the disclosure provides methods of reducing abnormal cell growth in a mammal in need thereof, comprising administering any of the compositions disclosed herein in combination with at least one adjuvant. In one 20 embodiment, the adjuvantis granulocyte machrophage colony-stimulating factor, The present disclosure further provides methods of reducing abnormal cell growth in a mammal in need thereof, comprising: a) providing any of the lyophilized compositions disclosed herein; b) adding water to the lyophilized composition to provide a reconstituted composition; and c) administering the reconstituted composition to said 25 mammal. The present disclosure further provides methods of reducing abnormal cell growth in a mammal in need thereof, comprising a) providing any of the lyophilized compositions disclosed herein; b) adding water to the lyophiized composition to provide a reconstituted composition; c) mixing the reconstituted composition with an adjuvant 30 compositions and d) administering the resulting mixture to said mammal. in onen embodiment, said adjuvantis granulytenischrophage colonyastimulating factor. The present disclosure further provides the use of any of the compositions disclosed herein r the manufacture of a medicament for the. treatment of abnormal cell growth in a mammal.

The present disclosure further provides processes for preparing a lyophilized composition comprising a KLH-peptide conugate, comprising subjecting any of the iquid compositions disclosed herein to a temperature of 420)$ or less, -30'C or less, 40"C or less, -50*C or lessor or less and to vacuum conditions 5 The present disclosure further provides processes of preparing liquid compositions comprising adding water to any of the lyophilized compositions disclosed herein. The present disclosure further provides methods for preparing a KLH-EGFRvIllI conjugate comprising: a) combining KLH with a inker and allowing the KLH and linker to 10 interact for a time ranging from 30 to 60 minutes- 35 to 55 minutes, 40 to 50 minutes, or 45 minutes and b) adding a peptide comprising, consisting essentially of, or consisting of SEQ ID NO: 1 or SEQ ID NO:2 to the activated KLH product resulting from step a) to provide the KLH-EGFRvIlli conjugate. In one embodiment step b) is carried out in a tangential flow filtration system, in a further embodiment, the retentate flow rate ranges 15 fror 10 to 1000 mL/min, for example 50 to 500 miL/in, for example 100 to 500 mULmin, and further for example, 50 mL/min, 100 mLimin, 150 mtUmin, 200 mUmin. 250 mUmin, 300 mUmin, 350 mUmin, 400 mUmin, 450 mUmin, or 500 mUmin. In one embodiment, the linker is combined with KLH in a linker;KLH molar ratio that ranges from 75:1 to 325:1, For example, in one embodiment, said linker:KLH molar 20 ratio ranges from 100:1 to 300:1, from 150:1 to 250:1, or 175:1 to 225:1. For example, in one embodiment said linkerIKLH molar ratio is 175:1, 180:1, 185:1, 190:1, 195:1, 200,1, 205:1 210:i 215:1, 220,1 or 2&. The present disclosure furher provides a liquid composition comprising a KLH EGFRvIAI conjugate, wherein after 26 weeks at 5*C, the number of particulates greater 25 than or equal to 10 pm as measured by USP <788> increased by less than 1000%: .ess than 750%, less than 500%, less than 400%, less than 300%, less than 200%, or oess than 100%. The present disclosure further provides a liquid composition comprising a KLH EGFRvIlI conjugate, wherein after 26 weeks at 5*C the number of particulates greater 30 than or equal to 25 pm, as measured by USP <788> increased by less than 1000%: .ess than 750%, less than 500%, less than 400%, less than 300%, less than 200%, or less than 100%.

Brief Description of the Drawings Figure 1 shows a general process schematic of one example of carrying out a KLH-peptide conjugation reaction, Figure 2A shows a summary of key operating parameters used during the 5 activation reaction of various cross-flow experiments Figure 2B shows a summary of key operating parameters used during the initial diafiitration step ir various cross-flow experiments. Figure 2C shows a summary of key operating parameters used during the conjugation step in various cross-flow experiments 10 Figures 3A 33, 3C and 3D show a summary of analytical results for crossflow filtration development experiments, Figures 4A, 4B, and 4C show a summary of reaction parameters that were used in Various experiments. Figure 5 shows a representative SEC HPLC chromatogram for the CDX1 10 15 KLHEGFRvIl conjugate showing the approximate retention times of the flee peaks, Figure 6 shows a plot indicating the effect of suif-SMCC ratio on eptope density Figure 7 shows a plot indicating the effect o activation tirne on epitope density. Figures 8A (dirmer) and 8B (monomer) show plots indicating the effect of sulfo SMOC ratio on SEC heterogenety. 20 Figures 9A (dimer) and 98 (monomer) show plots indicating the effect of activation time on SEC heterogeneity, Figure 10 shows a comparison of epitope density with linker slurry and linked dissolved in DMSO. Figures 1IA (dimner) and 113 (monomer) show plots indicating the effect of 25 DMSO on SEC profile of the activation reaction. Figures 12A and 12B show the statistcal analysis o epitope density results, indicating the interaction of sulfo-SMC:KLH ratio and protein concentration. Figures 13A and 13B show the statistical analysis of dimer results, indicating the interaction of sulfo-SMCC:KLH ratio and protein concentration 30 Figure 14 shows a summary of the reaction parameters used for the original and optimized process of preparing CDX-I10.

Detailed Description The present disclosure is based on the discovery of novelformulations for KLH peptide conjugates that provide beneficial properties, such as decreased pardculate and gel formation and increased stabAlity of the oonjugate: The formulations described 5 herein can also be lyophilized and reconstituted, providing additional benefits for shipping and long-termi storage. Such beneficial properties, and others that will be apparent to those of skill in the art, are described more fuly herein, Further, in accordance with the present disclosure, improved processes have been developed for the production of KLH-EGFRvII peptide conjugate vacccines. The new processes 10 disclosed herein provide optimized reaction conditions that are amenable to large-scale manufacturing protocols, and that result in improvements to the purity pro'e and control of key biophysical attributes, such as the ratio of peptides/KLH, and size heterogeneity In order that the present disclosure may be more readily understood, certain terms are first defined, Additional definitions are set forth throughout the detailed 15 description. Unless otherwise defined herein scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art, Further unless otherwise required by context, singular terms shall include pluralities and plural terms shail include the 20 singular, Generally, nomenclaturs used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art, The methods and techniques of the present disclosure are general performed 25 according to methods well known in the art and as descrbed in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated Such references include eg, Sambrook and Russell, foecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY (2001); Ausubel et al, Current Protocols in Molecuar Biology, John 30 Wiley & Sons, NY (2002) and Harlow and Lane Antibodies:A Laboratory ManualCold Spring Harbor Laboratory Press, Gold Spring Harbor, NY (1990) Enzymatic reactions and purification techniques are performed according to n manufacturer's specifications, as commonly accomplished in the art or as des"'ribed herein. The nomenclatures used in connection with, and the laboratory procedures and techniques o, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutica chemistry described herein are those well known and commonly used in the art, Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation1 formulation, and delivery, and treatment of patients. 5 As used herein, each of the following terms has the meaning associated with it in this section, The articles ' and 'an" are used herein to refer to one or to more than one (Ae, to at least one) of the grammatical object of the article. By way of example, 4'an element" means one element or more than one element, 10 As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Irnmunology-A Syrnhesis (2nd Edition, E, S. Golub and D, R. Gren, Eds., Sinauer Associates, Sunderland, Mass (1991)4 As used herein "KLH' refers to the protein Keyhole Limpet Hemocyanin, a multi subunit. oxygen-carrying, metalloprotein that is found in the hemolymph of keyhole 15 limpets (Megathura crenlata), or a fragment or subunit thereof. As used herein, the term "KLH-peptide conjugate" refers to a KLH protein, or a fragment or subunit thereof, that is covalentity bonded to a polypeptide that is unrelated to KLH. Such covalent bonding is typically accomplished through use of an appropriate chemical linker, 20 As used herein, the term "epitope de nsity% with reference to a KLH-peptide conjugate, refers to the average number of peptides that are coupled to each KLH subuatit Epitope density can be determined using amino acid composition analysis as described herein. The term "disaccharide as used herein, refers to a compound which, upon 25 hydrolysis, yields two monosaccharide molecules (e g. glucose, fructose, mannose, etc.). Suitable disaccharides include, but are not limited to, sucrose, lactose and trehalose, As used herein, the term "surfactant" refers to an excipient that can alter the surface tension of a liquid KLH-peptide conjugate composition. As discussed further 30 herein, examples of surfactants include, but are notlimited to, polysorbate surfactants poloxamers (e.g, poloxamer 18 and 407), triton surfactants, such as Triton X-100, and polysorbate surfactants such as Tween 20" and Tween 80 among many others that are commonly known to those of skill in the art.

The terms "yophilizationt "lyophilized" and "freeze-dried" refer to a process by which the material to be dried is first frozen and then the ice or frozen solvent is removed by sublimation in a vacuum environment As used herein, the term "EGFRvlil" refers to A peptide that is representative of 5 the variant mutant IlI that is present in epidermal growth factor receptor (EGFR) and typically comprises or consists of the amino acid sequence set forth as SEQ ID NO:1 or 2 or at least comprises the amino acid sequence "Lys Lys-GlyAsnyr" Abnormal cell growth", as used herein, unless otherwise indicated, refers to cell growth that is independent of normal regulatory mechanisms (e.g. loss of contact 10 inhibition), including the abnormal growth of normal cells and the growth of abnormal cells. This includes, but is not limited to the normal growth of; tumor cells (tumors) for example, mesothelioma, hepatobilliary (hepatic and biliary duct) a prirnary or secondary CINS tumor, a primary or secondary brain tumor, lung cancer (NSCLC and SCLC) bone cancer, pancreatic cancer. skin cancer, cancer of the head or neck, 15 cutaneous or intraocular melanoma, ovarian cancer, colon cancer, rectal cancer, cancer of the anal region stomach cancer, gastrointestinal (gastric coiorectal.and duodenal), breast cancer, uterine cancer, carcinoma of the faNlopian tubes, carcinoma a of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus cancer of the small intestine, cancer of the 20 endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, testicular cancer, chronic or acute leukemia chronic myeloid leukemia, lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis neoplasms of the central nervous system 25 (CNS), primary CNS lymphorna, non-Hodgkin's lymphorna, spinal axis tumors, brain stem gfioma, pituitary adenoma, adrenocortical cancer, gall bladder cancer, multiple myeloma, chdaniocarcinoma. fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one or more of the foregoing cancers. As used herein, the term ztreatingq or "treatment refers to both therapeutic 30 treatment and prophylactic or preventative measures,; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or conditions. Those in need of treatment include those already with the condition as well as those prone to have the condition or those in whom the condition is to be prevented, A "therapeutically effective amount" of a composition refers to an amount that is effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, which includes treatment or prophylactic prevention of abnormal cel growth, it is to benoted that dosage values may vary with the severity of the condition 5 to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual nee. and the professional judgment of the person administering or supervising the administration of the compositions and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition Likewise a 10 therapeutically effective amount of the composition may vary according to factors such as the disease state, ages sex, and weight of ithe individual, the ability of the therapeutic composition to elicit a desired response in the individual and the desired route of administration. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial 15 effects The term pharmaceuticall composition" refers to preparations which are in such form as to permit the biological activity of the active ingredients to be effective. "pharmceutically acceptable excipiernts" (vehicles, additives) are those, which can reasonably (.e_ safely) be administered to a subject to provide an effective dose of 20 the active ingredient employed. The term "excipient or carrierr" as used herein refers to an inert substance, which is commonly used as a diluent, vehicle, preservative, binder or stabilizing agent for drugs, As used herein, the term "diiuent refers to a pharmaceutically acceptable (safe and nontoxic for administration to a human) solvent and is useful for the preparation of the liquid forrmulations herein, Exemplary diluents 25 include, but are not limited to, sterile water and bacteriostatic water for injection (BWFi) Unless stated otherwise, the concentrations and pH values of the various components of the compositions disclosed herein are those concentrations at ambient conditions, (ie., at 25'C and atmospheric pressure) When concentration and pH agess are listed herein such ranges that are intermediate to the explicit ranges recited 30 are also intended to be part of the present disclosure For example, ranges of values using a combination of any of the values as upper and/or lower limits are intended to be included, in accordance with the present disclosure, it has been discovered that for a KLH peptide conjugate, the formation of particulates and/or gel can be reduced and the -~ 13~ stability of the conjugate can be improved by combining the conjugate with a potassium phosphate buffer, a disaccharide such as trehalose and a surfactant such as Tween 80. Futhermore, it has been discovered that when these components are used in certain amounts lyophilization (followed by reconstitution as an aqueous solution) is possible 5 while at the sare time minimizing particle and gel fomaation and maintaining the stability and biophysical characteristics of the conjugate. While riot wishing to be bound by any particular theory, it is believed that compositions of the present disclosure help to improve the stability and reduce particulate and/or gel formation of KLH-peptide conjugates by reducing the incidence of 10 one or more of the following; KLH-peptide aggregation, fragmentation oxidation freeze/thaw instability, instability associated wit shipping or transporting from one location to another, discoloration, and/or deamidation. Thus, the present disclosure provides KLH-peptide conjugate compositions having improved chenicaIl and/or physical stability as compared to previously disclosed compositions. 15 Therefore, in certain aspects, the present disclosure provides a composition comprising a KLH-peptide conjugate, a potassium phosphate buffer, a disaccharide, and a surfactant in still other aspects. the aforementioned compositions can h cdude additional pharmaceutically acceptable excipientsincluding, but not limited to. buffers sa ts antioxidants, tonicity agents, surfactnts, and mixtures thereof, Thus, the present 20 disclosure provides novel frmulations for KLH-peptide conjugates. KU-peptde corupates KLH is a complex multimeric protein that can be used as a carrier for antigenic peptides. Such peptides can be conjugated to KLH, and the resulting KLH-ppeptide 25 conjugate can be used as a vaccine in stimulating an immune response directed to the antigenic peptide, In one aspect of the disclosure, the antigenic pepide is conjugated to KLH by way of chemical cross-inking, typically using a beterooifunctional cross-inker. Several hetero-bifunctional crosslinkers are known in the art, In some embodiments, the 30 hetero-bifunctional crosslicker contairs a functional group which can react with first attachment sites, i'e. with the side-chain amino group of lysine residues of KLH, and a further functional group which can react with a preferred second attachment site ie a cysteine residue fused to the antigenic peptide and optionally also made available for reaction by reduction, The first step of the procedure, typically called the derivatization, s the reaction of the KLH with the cross4inker. The product of this reaction is an activated KLH, also called an activated carrier. In the second step, unreacted cross linker is removed using standard methods such as ge filtration or dialysis, In the third step, the antigenic peptide is reacted with the activated KIH, and this step is typically 5 called the coupling step, Unreacted antigenic peptide may be optionally removed in a fourth step, for example by dialysis. Several hetero-bifunctionalorosslinkers are known in the art. These include cross-linkers such as SIMPH Sulfo-MBS, Sulfo-EMCS Sulfo GMBS, Sulfo-SIAB, Sulfo-SMPB, Suffo-SMCC, SVSB SIA and other crosslinkers available, for example, from the Pierce Chemical Company (Rockford, IL, USA), and 10 having one functional group reactive towards amino groups and one functional group reactive toward cysteine residues. The above mentioned cross-linkers all lead to formation of a thioether linkage, One example of a KLH-peptide conjugate comprises a peptide known as EGFRII (SEQ ID NOI) that is chemically conjugated to the surface of the KLH protein. 15 This EGFRvilI peptide contains thirteen amino acids of a type il variant form of epidermal growth factor receptor (EGFRvilI) (see, e.g. U.S, Patent No, 5,401,828) The EGFRvll peptide can be coupled to primary amine groups (e.g. on Lys side chains) on the surface of KLH by including a Cys residue. on the N- or C-terminus of the peptide (see e g. SEQ ID NO;2 or 3), and then using sulfosuccinimidyk-4-(N 20 ma-oimidfmethyl)cylohexane-i1carboxylate (sulfo-SMCC) as a linker. Thus, a typical coupling reaction can be carried out as shown below in Scheme 1. + N / -- 1 Manttiudeictivated KU-I duIthSMCC KIIH-peptide ma opte Scheme I - 15 Those of skill in the art will recognize appropriate reaction conditions to achieve conjugation according to Sch eme 1. For example, the first step in Scheme 1 above (activation reaction) is where KLH, buffer, water, and linker are mixed together. This 5 activation reaction can then be allowed to proceed for approximately 45 5 minutes. At the end of the reaction time, the activation reaction mix can be diluted and transferred into an appropriate filtration system in order to remove excess linker and linker-related impurities, Peptide pre-dissolved in an appropriate buffer is then introduced and the conjugation reaction is allowed to proceed for 2 to 3 hours. At the end of the 10 conjugation0 process time, a second filtration operation can be performed in order to remove excess peptide and peptide-elated impurities and to buffer exchange into an appropriate final buffer, Further processing may include concentration adjustment by dilution, addition of excipients final filtering, and packaging, Figure 1 provides a general schematic of one example of how this process can be carried out, 15 The number of peptides per KLH subunit (also referred to herein as the "epitope density'), will depend on the specific reaction conditions, but typically ranges from about 30 to about 100 peptides per KLH molecule. This epitope density can be measured by comparison of total amino acid composition between conjugated and unconjugated KLH as described herein. The sizt distribution profile for KLH-peptide conjugates, such as 20 KLHEGFRvii conjugates, can include monomers, as well as multi-meric forms such as dimers, trirners, etc, Depending on the reaction conditions, this sie profile can vary and can be measured by analytical techniques such as size exclusion chronatography (SEQ. Where the peptide used in the above conjugation scheme is the EGFRvIll 25 peptide (as set forth in SEQ ID NO2 or 3), the resulting KLM conjugate is referred to as a KLH-EGFRvIl conjugate. An optimized process for producing a KLH-EGFRvIII conjugate was developed in order to address shortcomings of the original process while at the same time maintaining compatibility to drug substance lots which had already been in the clinic., The key analytical attributes that were. used for process development 30 were purity by anion-exchange (AEX) and reverse-phase HPLOC (RP-HPLC), molecular size distribution as measured by SEC HPLC, and epitope density. Criteria for an optimized process include maintaining or improving drug substance purity and producing a size distribution profile and epitope density within desired ranges.

Ant igenic pep)tides Polypeptides that are used for conjugation to KLH can be derived from natural sources and isolated from a mamnal such as, for example, a human, a primate, a cat, a dog, a horse, a mouse, or a rat. Polypeptides of the disclosure can thus be isolated 5 from cells or tissue sources using standard protein purification techniques Alternatively, polypeptides can be synthesized chemically or produced using recombinant DNA techniques For example, a polypeptide of the disclosure (e.g. those shown as SEQ ID NOs: I to 3) can be synthesized by solid phase procedures well known in the art, Suitable syntheses may be performed by utilising NT-boc or T-moc" 10 procedures. Cyclic peptides can be synthesised by solid phase methods employing the wellknown T-moc" procedure and polyamide resin in a fully automated apparatus: Alternatively, those skilled in the art will know the necessary laboratory procedures to perform the process manually Techniques and procedures for solid phase synthesis are described in Solid Phase Petide Syntheis: A Praetcai Approach by E Atherton 15 and R. C, Sheppard, published by IRL at Oxford University Press (1989) and Methods in MojaggCr Slooy Vol. 35: Peptide Synthesis Protocols fed, M W, Pennington and B. M Dunn) chapter 7, pp, 91-171 by L. Andreau et al. Alternatively a polynucleotide encoding a polypeptide of the disclosure can be introduced into an expression vector that can be expressed in a suitable expression 20 system using techniques well known in the art, followed by isolation or purification of the expressed polypeptide of interest. A variety of bacterial, yeast, plant, mammalian, and insect expression systems are available in the art and any such expression system can be used Optionally, a polynucleotide encoding a polypeptide of the disclosure can be translated in a cell-free translation system. 25 Keyhole Limpet Hemocyanin Those skilled in the art recognize KLH as a beneficia carrier of antigenic peptides, due to its ability to increase the immune response because of its large size and distinct epitopes (see, e.g. Harris et al., M/cron 30(6);597-23 (1999)), KLH is 30 typically obtained directly by purification from keyhole limpets, and is commercially available from several sources (e.g Thermo Scientific, Biosyrn) Prepaation of KLH-e ptide Coniugate Con positions The KLH-peptide conjugates of the present disclosure are typical formulated as pharmaceutical compositions for parenteral administration to a subject In one embodiment, the composition is a liquid compostion in another embodiment, the 5 coniposition is a Iyophilized composition, The compositions of the present disclosure comprise a KLH-peptide conjugate, such as KLH-EGFRvlWi conjugates, combined with a potassium phosphate buffer, a dissacharide, and a surfactant. The amount of KLH~ peptide conjugate. such as a KLH-EGFRvlsl coniugate, can be present in the compositions of the present disclosure in an amount of from 0.01 to 20.0 mg/mL, for 10 example 0.1 to 10 mg/mL, or 05 to 5 mg/mL. For example, in particular embodiments, the KLH-peptide conjugate, such as a KLH-EGFRv'lI conjugate, is present in a liquid formulation in an amount of from 0(5 to 1.5 mg/rn L such as 1,0 mg/mL, Such compositions can be aqueous liquid compositions with a pH range from 7 to 8, In a particular embodiment, the pH is from 7,3 to 75, such as 7.4. Those of skill in 15 the art will appreciate that there will be some inter-apparatus variabilty in the measurement of the pH of any particular solution, typically by as much as 01 pH units, Accordingly, where pH numbers are recited throughout the present disclosure, one of skil in the art wil recognize that such numbers are intended to encompass such inter apparatus variability. 20 The compositions of the present disclosure also include lyophilized compositions that have subsequently been reconsituted by the addition of solvent, such as water, The compositions of the present disclosure can include a potassium phosphate buffer, which is typically used to adjust the pH to a desired level. However, the. present disclosure is not intended to be limited by any specific mechanism by which the 25 potassium phosphate buffer acts in the compositions disclosed herein, and the potassium phosphate buffers used in the composition dislcosed herein may achieve their properties through mechanisms that are altogether unrelated to their ability to adjust the pH to a desired level As used herein, the term "buffer" refers to an added composition that allows a liquid composition to resist changes in pH, in certain 30 embodiments the added buffer aTows a liquid KLH-peptide conjugate composition to resist changes in pH by the action of its acid-base conjugate components When a concentration of a buffer is referred to, it is intended that the recited concentration represent the molar concentration olfthe free acid or free base form of the buffer Although those of skill in the art will appreciate that phosphate buffers are typically not used in parenteral formulations especially in lyophilized products since phosphate salts have a tendency to crystallize during freezing - the present disclosure surpisingly shows that potassium phosphate buffer is useful in KLH-peptide conjugate 5 formulations without any such crystallization, in particular, potassium phoshate buffer can be used in a concentration that ranges from 5 mM to 15 nM, for example from 7 mM to 12 mM, from 8 to II mv, or 9 5 mlM to 10.5 mM, Those or skii in the art will recognize that potassium phosphate buffer is typical prepared as a mixture of monobasic dihydrogen phosphate and dibasic monohydirogen phoshate (eg, KH 2

PO

4 10 K2HPO 4 ) in sufficient respective concentrations to arrive at the desired pH. Such buffers are commercially available, or those skilled in the art will recognize how to prepare and use such buffers within the context of the present disclosure, The compositions of the present disclosure can also comprise a disaccharide, such as trehalose, for use as a cryoprotectant during freezing and as a lyoprotectant 15 during freeze-drying, However, the present disclosure is not intended to be limited by any specific mechanism by which the disaccharide acts in the compositions disclosed herein, and the disaccharides used in the compositions disicosed herein may achieve their properties through mechanisms that are altogether unrelated to their abiity to act as a cryoprotectant dunng freezing and as a lyoprotectant during freeze-dryikg, In the 20 present disclosure, such a disaccharide can be used in an amount of from 70 to 110 mg/mL Those skilled in the art will recognize that disaccharides, such as trehalose; dextran, and sucrose (among others) are readily available through commercial sources. The composition of the present disclosure can also comprise a surfactant, such as polvsorbate 80, to help decrease particle formation by decreasing the interaction of 25 KLH-peptide conjugates with air-water or ice-water interfaces. However, the present disclosure is not intended to be limited by any specific mechanism by which the surfactant acts in the compositions disclosed herein, and the surfactants used in the compositions dislcosed herein may achieve their properties through mechanisms that are altogether unrelated to their ability to decrease the interaction of KLH-peptide 30 conjugates with air-water or ice-water interfaces. As used herein, the term "surfactant" refers to an excipient that can alter the surface tension of a liquid KU--peptide conjugate comriposiion. In certain embodiments, the surfactant redu liquid KLH-peptide conjugate composition, In still other embodiments, the *surfactant may contribute to an improvement in stabiity of a KLH-peptide conjugate. such as a KLH-EGFRvlii conjugate. For example, the surfactant may reduce agregation of the KLHpeptide conjugate and/or minimize the formation of particulates in the composition and/or reduce adsorption. The surfactant may also improve stability of the KLH-peptide conjugate during and after a freeze/thaw cycle. 5 Suitable surfactants include polysorbate surfactants. poloxamers e g poloxamer 18 and 407), triton surfactants, such as Triton X-100', polysorbate surfactants such as Teen 20 and Tween 80 sodium dodecyl sulfate, sodium laurel sulfate. sodium octil glycoside, laury sulfobetaine myristyhsulfobetaine linoleylsulfobetaine stearyl~ sulfobetaine siavylsarcosine, myristylsarcosine inoleylsarcosine, stearyl-sarcosine 10 linoleyl-betaine, myristyl-betaine, cety- betaine, lauroamidopropyl-betaine, cocamidopropyeiaine linoIeamidopropyI-b etaine, myristamidopropyl-betaine pal midopropyl-betaine, isostearam idopropyl-betain e, myristamidopropyldimethylamine pal midopropydirnethylamine isostearamidopropyl- dimethylamine, sodium methyl cocoyl-taurate, disodium methyl oleyktaurate, dihydroxypropyl peg 5 linoleammonium 15 chloride, polyethylene glycol, polypropylene glycol, and mixtures thereof. In one embodiment, the surfactant is a polysorbate surfactant conprising at least one excipient that is selected from the group consisting of poiysorbate 20, polysorbate 21, polysorbate 40, polysortate 60, polysorbate 61, polysorbate 65, polysorbate 80. polysorbate 81, polysorbate 85, and mixtures thereof, 20 The concentration of the surfactant when present in the composition generally ranges from 0.01 mg/mL to 10 mg/mL, from 0,05 mg'mL to 5.0 mg/mL, from 0,1 mg/tl to 1.0 mg/mt, or from 02 mg/mL to 0.7 mg/mL. In another embodiment, the surfactant is present in an amount that is 0.2n m/mL In another embodiment, the surfactant is present in an amount that is 0.5 mg/mtL In another embodiment the sUrfactant is 25 polysorbate 80 (e g, Tween 80*y, and is present in an amount from 01 to 0.3 mg/mL, for example 0,2 mg/mL, Ranges intermediate to the above-recited surfactant concentrations are also intended to be part of this disclosure. For example, ranges of values using a combination of any of the above- recited values as upper and/or lower limits are intended to be included, Those skiled in the art will recognize that surfactants. 30 such as Tween 8 0 h are readily avaiable through commercial sources. Once -a KILH-peptide conjugate, such as a KLHiGFRvtII conjugate, is maded can be formulated with the components as described above using methods that are well known to those of skill in the art, For example, a KLH-EGFRvlil conjugate can be introduced into a suitable compounding vessel and stirred gently to achieve homongenvety. The conjugate can then be diluted to about 1.0 mg/mL with the formulation buffer (esg. potassium phosphate). The solution can then be sampled to confirm the appropriate pH and concentration. The solution can then be passed through two sterillzing filters (0,22 micron) in series 5 Routes of Adcministration and Dosages The compositions of the present disclosure may be in liquid soutions (e.-g. injectable and infusible solutions) The preferred form may depend on the intended mode of administration and therapeutic application. Typical preferred compositions are 10 in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans. The preferred mode of administration is parenteral (e g., intravenous, intradermal, intramarterial, subcutaneous intraosseous, intraperitoneat intramuscular, and intrasternally) or by infusion techniques, in the form of sterile injectable liquid or olagenous suspensions. As will be appreciated by the 15 skilled artisan, the route and/or mode of administration will vary depending upon the desired results In one embodiment, the KLH-peptide conjugate composition is administered by intravenous infusion or injection. in another embodiment, the composition is administered by intranuscular or subcutaneous injection. Therapeutic compositions 20 typically are sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, or liposome Sterile injectable solutions can be prepared by incorporating the KLH-peptide conjugate composition of the required amount in an appropriate diluent with one or a combination of ingredients enumerated above, as required, followed by sterilization (e.g., fitter 25 sterilization) Generally dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medum and the required other ingredients from those enumerated above. The sterle injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a 30 solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water Ringer's solution and isotonic sodium chloride solution. in addition, stehrle, fixed oils are conventionally employed as a solvent or suspending mediumr For this purpose, any band fixed oil may be employed, including synthetic mono~ or diglyoerides, In addition n-3 polyunsaturated fatty acids may find use in the preparation of injectabies In the case of sterile powders for the preparation of sterie injectable solutions, the preferred methods of preparation are vacuum drying, spray-drying, and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidAy of a 5 solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorptior. for examplemonostearate 10 salts and gelatin or by formulating the composition into prolonged absorption forms such as, depots' liposomes, polymeric microspheres, polymeric gels, and implants Other methods for admnstration of the compositions described herein include dermal patches that release the medications directly into a subject-s skin. Such patches can contain the compositions of the present disclosure in an optionally buffered, liquid 15 solution dissolved and/or dispersed in an adhesive., or dispersed in a polymer Stili other methods for administration of the compositions described herein include liquid ophthaimological drops for the eyes. The KLHbpeptide conjugate composition may be adminstered once, or mwtiple times For example, the cornpostion may be administered from once daily to |once 20 every six months or longer. The administering may be on a schedule such as three times daily, twice daily, once daily, once every two days, once every three days, once weekly, once every two weeks, once every month, once every two months, once every three months and once every six months, The composition may also be administered continuously via a miripump. The 25 composition may be administered at the site of the tumor or inflamed body part, into the tumor or inflamed body part or at a site distant fromthe site of the tumor or inflamed body part The composition may be administered once at least twice or for at least the period of time until the condition is treated, palliated or cured. The KLIHpeptide conjugate composition generally may be administered for as long as the tumor is 30 present provided that the composition results in the desired therapeutic effect The composition typically would be administered as part of ab parmaceutical compositon as described herein. The compositions of the disclosure may include a therapeutically effective amount of a KltH-peptide conjugate as disclosed herein, In preparing the formulation, the therapeutically effective amount of the KLH-peptide conjugate present in the formulation can be determined, for example, by taking into account the desired dose volumes and models) of administration, the nature and severity of the condition to be treated, and the age and size of the subject Exenipary, nonilmiting dose ranges for administration of the compositions of the 5 present disclosure to a subject are from 0,01 mg/kg to 200 ng/kg (expressed in terms of milligrams (im) of KLH-peptide conjugate administered per kilogram (kg) of subject weight), from 01 mg/kg to 100 mg/kg, from 1.0 mg/kg to 50 mg/kg, from 5.0 mg/kg to 20 mg/kg, or 15 mg/kg. For purposes of the present disclosure an average human subject weighs about 70 kg Ranges intermediate to any of the dosages cited herein, eg 0 01 10 mg/kg - 199 mg/kg, are also intended to be part of this disclosure, For example, ranges of values using a combination of any of the recited values as upper and/Or lower limits are intended to be included. Dosage regimens can also be adjusted to provide the optimum desired response (e.g.. a therapeutic or prophylactic response) by administering several divided doses to 15 a subject over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. it is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a 20 predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier, The specification for the dosage unit forms of the invention are dictated by arid directly dependent on (a) the unique characteristics of the KLH-peptide conjugate and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations 25 inherent in the art of compounding such KUHpeptide conjugate for the treatment of sensitivity individuals The liquid formulations of the present disclosure can be prepared as unit dosage forms.. For example a unit dosage per vial may contain from I to 1000 milliliters (mL) of different concentrations of a KLH-peptide conjugate, In other embodiments, a unit 30 dosage per vial may contain 0.2 niL, 0.4 mL, 06 moL, 0,8 nL, I mL, 2 mL 3 mL. 4 mL. 5 mL 6 mi 7 mL, 8 mL, 9 nL, 10 nil 15 nL, 20 nL, 30 mL, 40 m, 50 nL or 100 iL of different concentrations of a KLH-peptide conjugate, If necessary, these preparations can be adjusted to a desired concentration by adding a stenle diluent to each vial. The .iquid formulations of the present disclosure can also be prepared as unit dosage forns in sterile bags, containers pre-filled syringes, pens, r microneedles which can be used for direct administration and/or are suitable for connection to an intravenous administration line or catheter, KEH-peptide conjugates, such as KLiH--EGFRvli conjugates, can be combined 5 with other immunostimuatory agents, such Is granulocyte macrophage colony stimulating factor (GM-CSF), e g. sargraniostini or molgramostim For example, GM CSF can be combined directly with a KLH-EGFRvll conjugate, and then administered to a subject, In one. example, 0,35 mL of GM-CSF that is concentrated at 0,5 mg/mi can be combined with 0.6 imL of a KLH-EGFRvi conjugate composition that is concentrated 10 at 1 mg/mL to provide 0.95 mL of combined solution containing 0.5 mg of a KLH EGFRvl conjugate and 0.15 mg of GM-CSF. This combined composition can then be administered directly to a subject. Those skilled in the art will recognize that a variety of amounts and concentrations can be used to provide the apporate dosage amount lo a subject in need thereof. 15 Stabiliy Assessment The present disclosure provides stable liquid and lyophilized compositions comprising a KLH-peptide conjugate as described herein. A stable composition is desirable to maintain or resist changes in for example, product appearance and 20 integrity (including physical or chemical degradation potentially leading to a reduction in biological activity) Various analytical techniques and indicators for measuring protein stability are reported in the literature and a number of these techniques and indicators are reviewed ir Peptide and Proteir Drug Delivery, 247301, Vincent Lee Ed., Marcel Dekker Inc- New York, NY., Pubs. (1991) and Jones, A., Adv. Drug Delivery Rev. 10' 25 29-90 (1993). In general, the compositions of the present disclosure exhibit improved stability when subjected to low storage temperatures over a period of time, and/or when subjected to one or more freeze/thaw cycles. The stability of KLH-peptide conjugates can be measured using a variety of analytical techniques that are well known to those of 30 skill in the art For example, analytical assays such as appearance (as measured by visual inspection andoor by UV light scattering) microscopy, light obscuation, size exdl fusion chromatography, anion exchange (AEX) chromatography; and reveraephase high -pressure liquid chromatography, Such assays can provide data on stability for multiple degradation pthways, such as change in content of monomer/dirner ratio, formation of peptidelinker impurities, change in A280/A340 ratio, and AEX retention time of a KLH-peptide conjugate peak due to formation of acidic species Such analytical assays can be used to monitor the stability of a conjugate as a function of time and temperature. 5 Protein fragmentation in a liquid pharmaceutical composition can be measured by various methods known in the art, Such methods include for example, size exclusion chromatography, ultraviolet detection e g., at 214 nanometers), SDS-PAGE and/or matrix- assisted laser desorption ionizaion/tim e-of-flight mass spectrometry (MALDI/TOF MS). Protein fragmentation resulting in a charge alteration (e g., occurring 10 as a result of deamidation) can be evaluated, for example, by ion-exchange chromatography or isoelectric focusing (EF) Composition discoloration general can be measured by visual observation of the composition itself The compositions disclosed herein generally reduce compostion discoloration (e.g,. pink or yellow) and/or maintain composition clarity (e g, turbidity, cloudiness and/or particulate formation), For 15 purposes of the present disclosure the term "discoloration* refers to both changes in color (e.g, ftom clear and colorless to pink or yellow) and to changes in clarity (cxg. from clear and colorless to turbid, cloudy and/or having particulates). Composition discoloration general can be measured using additional techniques such as by ultraviolet detection at 214 nanometers and/or by visual comparison against a standard 20 color scale of the compositions being compared As used herein, the term "a freezelthaw cycle" refers to techniques for using a liquid sample after frozen storage, wherein the temperature of the sample is lowered to a temperature of &C or lower in order to freeze the liquid sample, and ther-subjecting the sample to a temperature which will restore its liquid state for a sufficient period of 25 time to permit use of the sample, followed by and return to frozen storage, preferably at a temperature of 0'C or lower. As used herein, the term "frozen storage" refers to freezing and maintaining a previously liquid sample at a temperature of OTC or below, and preferably -20"C or lower, 30 Methoos of Treatment Any of the KLH-peptide compositions described herein may be used therapeutically. in one embodiment, the KLH-peptide conjugate is a KLH-EGFRI conjugate, as described herein, and is used to treat a human subject, Alternatively, the subject may be a mammal that expresses the EGFRvII variant, Thus, the KLH- EGFRvIll conjugate compositions disclosed herein may be adm-inistered to a non human mammal expressing the EGFRvll variant for veterinary purposes or as an animal model of human disease. Such animal models may be usef for evaluating the therapeutic efficacy of conm positions of the present disclosure. 5 The present disclosure provides methods of reducing abnormal cell growth in a mammal in need thereof, comprising the step of administering to said mammal any of the compositions disclosed here, particularly those wherein the KLH-peptide conjugate .is a KLHG-EFRvIlI conjugate, In one embodiment, the abnormal cell growth is cancerous In a further embodiment, the cancer is Olioblastoma, The present 10 disclosure further provides methods of reducing abnormal cell growth in a mammal in need thereof, comprising the steps of a) mixing any of the compositions as decribed herein with an adjuvant; and b) administering to said mammal the resulting mixture, In one embodiment, the KLH-peptide conjugate is a KLH-EGFRviI conjugate, and the adjuvant is granulocyte macrophage colony-stimuating factor (GM-CSF), e.g. 15 sargram ostim or molgramostim. The present disclosure further provides methods of reducing abnormal cell growth in a mammal in need thereof, comprising: a) providing any of the lyophilized compositions disclosed herein, adding water to the lyophilized composition to provide a reconstituted composition, and administering the reconstituted composition to said 20 mammal. Also provided herein are methods of reducing abnormal cell growth in a mammal in need thereof, comprising providing any of the lyophilized compositions as described herein, adding water to the Iyophilized composition to provide a reconstituted composition, mixing the reconstituted composition with an adjuvant composion, and administering the resulting mixture to said mammal. In particular embodiments, the 25 mammal is human, and the KLH-peptide conjugate is a KLH-EGFRvli conjugate. Therapeutc methods involve adminis tenng to a subject in need of treatment a therapeutically effective amount, or "effective amount", of a composition comprising a KLHpeptide conjugate, such as a KLH -EGFRvid conjugate, as contemplated by the present disclosure. As used herein, a therapeutically effective", or "effective amount 30 refers to an amount of a KLH-peptide conjugate thereof that is of sufficient quantity to ret in a decrease in severity of disease symptoms, an increase in frequency and duration of disease sympton-free periods, or a prevention of impairment or disability due to the disease affliction - either as a single dose or according to a multiple dose regimen, alone or in combination with other agents, One of ordinary silI in the art would be able to determine such amounts based on such factors as the subjects size, the severity of the subjects symptoms and the particular conposition or route of administration selected. The subject may be a human or non-human animal (e,.g rabbit, rat, mouse, rnonkey or other lower-order primate) A composition of the present 5 disclosure might also be co-administered with known medicaments, The KLH-EGFRvill conjugate compositions disclosed herein can be used as a therapeutic product in a variety of situations where the EGFRvI variant is undesirably expressed or found, Given the expression of the EGFRvil variant in various cancers, such as glioblastonia, disorders and conditions particularly suitable for treatment with a 10 KLH-EGFRviI conjugate compositions of the present disclosure include abnormal cell growth, for example, mesothelioma hepatob iary (hepatic and billiary duct), a primary or secondary CNS tumor, a primary or secondary brain tumor, lung cancer (NSOLC and SCLC), bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, ovarian cancer, colon cancer, rectal cancer, cancer 15 of the anal region, stomach cancer, gastrointestinal (gastric colorectaland duodenal), bJreast cancer, uterine cancer, carcinoma of the fallopian tubes, arinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer 20 of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, testicular cancer chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphornas, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma; carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, non-Hodgkin's lymphoma, spinal axis tumors, brain 25 stem glioma pituitary adenoma, adrenocortical cancer, gall bladder cancer, multiple myeloma, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma or a combination of one or more of the foregoing cancers. The compositions described herein can be administered by any suitable means, which can vary, depending on the type of disorder being treated. Possible 30 administration routes include parenteral (e.g., intravenous, iradermal, intra-arterial, subcutaneous, intraosseous intraperitoneal, intramuscuiar, and irrastemally intrapulmonary and intranasal, and, if desired for local immunosuppressive treatment or Intralesional administration. In addition, compositions of the disclosure minight be administered by pulse infusion, with, e.g., declining doses of the KL H-peptide conjugate.

Preferably, the dosing is given by injections, most preferably intradermal or .ntramuscuiar injections. depending in part on wh ether the administration is brief or Chronic. The amount to be administered will depend on a variety of factors such as the 5 clinical symptoms, weight of the individual, whether other drugs are administered The skilled artisan will recognize that the route of administration will vary depending on the disorder or condition to be treated, For example, determining a therapeutically effective amount of a composition comprising a KLH-peptide conjugate (eg. a KLH-EGFRvIll conjugate) according to the present disclosure will largely depend on particular patient 10 characteristics, route of administration, and the nature of the disorder being treated, General guidance can be found, for example, in the publications of the international Conference on Harmonization and in Remington's Pharmaceutica Sciences, chapters 27 and 28, pp, 484-528 (18th ed., Alfonso R. Gennaro Ed Easton, Pa: Mack Pub. Co., 1990) More specifically, determining a therapeutically effective amount will depend or, 15 such factors as toxicity and efficacy of the medicament, Toxicity may be determined using methods well known in the art and found in the foregoing references. Efficacy may be determined utilizing the same guidance, Adjuvants 20 In some embodiments, a KLH-peptide conjugate composition of the present disclosure (esg. a KLH-EGFRvli conjugate) can be combined with or administered concurrently with, at least one adjuvant, Suitabe adjuvants include those suitable for use in mammals, preferably in humans. Examples of known suitable adjuvanis that can be used in humans include but are not necessarily limited to, alum. aluminum 25 phosphate, aluminum hydroxide, MF594 (43% w squalene, 0.5% w/v polysorbate 80 (Tween 80), 0.5% wiv sorbitan trioleate (Span 85)), G-containig nucleic acids (where the cytosine is unmethylated), QS21 (saponin adjuvant), MPL (Monophosphoryi Lipid AL 3DMPL (30deacylated MPL). extracts from Aquila. ISCOMS (see, e.g., Sjblander et a'.. J Leukocyte Bioh 642713 (1998); PCT Publication Nos VO 90103184, 30 WO 96/11711 WO 00/486305 WO 98/36772, WO 00/41720, WO 06134423 and WO 07/026190). LTICT mutants, poly(D Liactide-coglycolide) (PLG) ricropartidesQuil A, interleukins. and the like. For veterinary applications including but not limited to animal experimentation, one can use Freund's, N-acetyl-muramyL-threonyl-D-isoglutamine (thr-MDP), N-acetyi-normummylLalanyi-D-isoglutamine (CGP 11637, referred to as nioriMDP) N-acetylmuramyi-Lalanyi-DAssaglutaminyV L-alanine-2}(1 t2Ldipalmiitolusn glycero3-hy droxyphosphoryloxybrethylamine (CGP 19835A, referred to as MTP-PE), and RIB which contains three components extracted from bacteria monophosphoryl pid trehose dimycolate and cell wal skeleton (MPL+TDM+CWS) in a 2% 5 squalneTween 80 emulson, Further exemplary adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) olkr-water emulsion formulatons (with or without other specific im munostimulating agents such as muramy peptides (see below) or bacterial cell wall components) such as for example (a) MF59" (PCT Publication No 10 WO 9014837; Chapter 10 in Vaccine design: the subunit and adluvant approach eds. Powell & Newman, Plenum Press 1995), containing 5% Squalene, 0,5% Tween 80 (polyoxyethylene sorbitan monooleate), and 0-5% Span 85 (sorbitan trioleate) (optionally containing muramyl tri-peptide covalently linked to dipaimitoyl phosphatidylethanolamine (MTP-PE)) formulated into submicron particles using a 15 microfluidizer (b) SAF, containing 10% Squalene, 04% Tween 80, 5% pluronic-.blocked polymer L121, and thrMDP either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) RIBI adjuvant system (RAS), (Ribi Imm unoch em, Hamilton, MT) containing 2% Squalene 0.2% Tween 80, and one or more bacterial cell wall components such as monophosphorylipid A (MPL) trehalose 20 dimycolate (TDM), and cell wall skeleton (GWS), preferably MPL + CWS (DETOXT); (2) saponin adjuvants, such as Q021, STIMULON

T

', (Cambridge Bioscience. Worcester, MA), Abisco@ (isconova, Sweden), or Iscomatrix@ (Commonwealth Serum Laboratories, Austaila) may be used or particles generated therefrom such as ISCOMs (imrunostimulating complexes), which ISCOMS may be devoid of additional detergent 25 e-g; PCT Publication No. WO 00/07621; (3) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (iFA); (4) cytokines, such as interleukins (eg ILl, 1L2, IL4 IL-5, L-6, IL-7 L~12 (,PCT Publication No. WO 99/44636), etc. interferons (exg gamma inerferon), qranulocyte-macrophage. colony stimulating factor (GM~CSF, such as moigramostim or sargramostim), tumor necrosis factor (TNFx etc.; (5) 30 monophosphoryl lipid A (MPL) or 3-0-deacylated MPL (3dMPL) eg. Great Britain Patent No. GB-2220221 and European Paten No. EP-A~089451, optionally in the substantial absence of alum when used with pneumococcal saccharides eg, PCT Publication No, WO 00/56358, (6) combinatons of 3dMPL with, for example, 0S21 and/or oil-in-water emulsions eg; EP-A-0835318, EP-A-0735898, EP~A-0761231; (7) oligonucleotides comprsing CpG motifs [Krieg, Vaccine (2000) 1961&822; Krieg Curr Opin Mot Ther (2001) 315-24. Roman et al, Nat Med, (1997) 3:849 -854; Weiner et al, PAS USA (1997) 94;10833-10837; Davis et at J. Irnmunol (1998) 1 6:870-876; Chu et al, J Exp4Med (1997) 186;1623-1631; Lpford ata Ear . immunot, (1997) 27:2340 5 2344; Moldoveami el aL Vaccine (1988) 16,12164224, Kdeg et aL Nature (1995) 374:546-549; Klinman et ta PNA S USA (1996) 93:2879-2883; Ballas et al J, immunot (1996) 157:1840-1845 Cowdery et al Immunel (1996) 156:4570-4575 Halpern a! at Cell ImmunoLi (1996) 167 72-78; Yamamoto et at din J Cancer R (988) 79:866~ 873 Stacey et a4 i hmmuno L (1996) 157-21 16-21 22; Messina et a4 J, mmunol, (1991) 10 147:1759-1764; Yi et a J immun)l (1996) 157:4918-4925 Yi a .Jtmmunol (1996) 157:5394-5402; Yi et al J. hmmunet (1998) 160:4755.-4761; and Yi et al J Immunot (1998) 160:5898~5906; PCT Publication Nos. WO 96/02555, WO 98W16247; WO 98118810, WO 98t40100, WO 98/55495, WO 98/37919 and WO 98/52581j ia; containing at least one CG dinucleotide, where the cytosine is unmethylated (8) a 15 polyoxyethylene ether or a polyoxyethylene ester e-g; PCT Publcation No. WO 99/52549; (9) a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol (PCT Publication No. WO 01/21207) or a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-onic surfactant such as an octoxynol (PCT Publication No, WO 01/21152); (10) a saponin and an 20 imminostimulatory oligonucleotide (e.g, a CpG oligonucleotide) (PCT Publication No. WO 00/62800); (11) an immunostimulant and a particle of metal salt e.g. P-CT PublicatIon No, WO 00/23105; (12) a saponin and an oil-in-water emulsion e.g. PcT Publication No, WO 99/11241; (13) a saponin {eSg. Q821) + 3dMPL + IM2 (optionally + a stem) -a PCT Publication No, WO 98/57659 (14) other substances that act as 25 immunostimulating agents to enhance the efficacy of the composition, such as Muramyl peptides including N-acetyimuramylL-threonylDisoglutamine (thr-4DP), N-25 acetyl norm uramyl-L-alanyl-D-isoglutam ine (nor-MDP, N-acetylmuramyL-aanylD isoglutarninyl-L-alanine-2-(1 -- dipalmitoylsn-glycc-ro--3-hydroxyphosphoryoxy) ethylamine MTP-PE), (15) ligands for tol-ike receptors (TLR), natural or synthesized 30 (e~g, as described i-i Kanzler et al Nature M Ia 13:15522-1559 (2007)); including TLR3 ligands such as polyC and similar compounds such as Hiltonol and Ampligen. In one embodiment, a composition of the present disclosure is administered concurrently with or is combined with prior to administration at east one adjuvant, In a - 30 particular embodiment, said adjuvant is a cytokine, in particular GMICSF (e g. molgramostirm or sargramostiri). Adi/c/es of Manufacture 5 In another embodiment of the disclosure, an article of manufacture is provided comprising a container, which holds the liquid composition composing a 'KLH-peptide conjugate as described herein, and. optionally provides instructions for its use Suitable containers include for example, bottles, vials, bags and syringes. The container may be formed from a variety of materials such as glass or plastic. An exemplary container is a 10 3-20 cc single use glass vial. Alternatively, for a multidose formulatdon, the container may be a 3-100 cc glass vial, The container holds the composition the the label on, or associated with, the container may indicate directions for use, The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers diluents. filters, needles, syringes, and package 15 inserts with instructions for use Contraindications, and/or lists of potential sidereffects. The present disclosure also provides a kit for preparing a liquid composition of a KLH-peptide conjugate comprising a first container, comprising a KLH-peptide conjugate, such as a KI-H-EGFRvll conjugate, in solution, and a second container comprising a sufficient amount of an adiuvant, such as GM-CSF, alone or in 20 combination with other excipients. The present disclosure is further illustrated by the following examples which should not be construed as further limiting. The contents of all figures and all references, patents and published patent applications cited throughout this disclosure are expressly incorporated herein by reference in their entirety. 25 EXAMPLES In the examples that follow, :epitope density" was determined using amino acid analysis as follows, The couple of the Ef3FRvll peptide to KLH via a bifunctional inker leads to a distribution of peptides conjugated to the KLH carrier protein. The 30 number of peptides conjugated to KLH was measured using amino acid composition analysis. There are several amino acids whose contribution comes exclusively frorn the KLH carrier protein and not the peptide, By comparing those amino acids to those that originate from both the peptide and KLH, the molar excess can be calculated. The molar excess of these amino acids can be attributed to the EGFRvIll peptide and is used to calculate the number of peptides per KLH subunit. Prior to amino acid analysis, the samples are buffer exchanged to remove any residual process impurities such as peptide dirner or peptide liner conjugate. These impurities have the potential to 5 contribute to the amino acid levels and skew the results, The samples are then hydrolyzed using 6N HCl, labeled with a fluorescent tag, a separated and quantified using HPLC. Example . Effect of pH/buffer 10 The objective of the study was to investigate effect of pH and buffer species on the storage stabifty of CDX-1 10 at different temperatures (5), 25C and 40"C) for both liquid and lyophiized forrmuiations (Table IA). The pH range expIored was 45 l.o 7.4, and the different buffering species were Potassium Phosphate, Sodium Phosphate, Succinate and Histidine. The data reported here is at the end of 12 weeks for all 15 formulations except for succinate (J) that was stopped at the end of 8 weeks; Protein concentration, pH and polysorbate content were kept constant as shown in Table IA. liq" indicates a liquid formlation, whereas yo" dicates alyophilized formulation. The pH ranges were chosen for intradermal administration, All comparisons are made agmnst the current formulation F (both liquid and lyo ii PBS; composition in Table 1)Q 20 Table IA: Formulation composition for pH/buffer study Fomuato K- K6 66 6 6 siq) (s (ia 2 (ye) (IM lye) (14 @iOl ON) lye) (1 ) mM Histidim ET--- - - I 1i "4 '- I Trehalose 7F 56 7 ::0' '.2 32 oPBS copostion given in Tab 1B. Formulation was studi for 8 weeks only. Formulations C and D differ only in trehalose content by 6 mg/mL All lyophilized formulations were reconstituted with WRI such that final CDX110 concentration was 5 -1 .0mg/m Table 1S: Current CDX-110 formulation composition in PBS (F) Componn Concentration Ampr mtds d OI mg/mL __ _____________ mg e . nLds Phosphate buffer 10 rmM 0.72 mg Na2HPC4 Sodium chloride )3 mM 4m PoltssimChoride 2. M 0. mg 10 Preparation of Buffer Solutions Five buffer solutions were prepared as described in Table 1C below Each solution was prepared by first dissoling an amount of the buffer species (isted in fable 15 1 wterfor injection (WFI) The pH of each buffer solution was measured. The bufrfer solution was then filtered through a sterilization filter (0.22 micron pore size) into a sterilized receptacle for subsequent use. Table 1C: Cuffer Solutions 5 iLTY H____Buffier 8 peie BufrStrengt (og) Histicdine 6.5 LHistidine 15 L-Hist dine HCI monop hydrate Succinate 14 5 Succinic acid 10 ____________ Sodium Succinate __________ PBS 74 Sodium phosphate basic, 10 phosphate anhydrous buffer saline) Potassium phosphate monobasic Sodium chloride ______ Potassium chloride __________ Potassium 74 Potassium phosphate dibasic 10 Phosphate anhydrous _________ otassiug hatehg monobasic __________ Sodium 7 4 Sodium Phosphate. Monobasic 10 - 33 Buffer TypJ _pH Buffer pecles Buffer Strength (mM)_ phosphatenohydrate Sodiumr Phosphate, Dibasic, Preparation of CDX- 110 Formulation The formulations That were evaluated are listed in Table 1A. To prepare each formulation, CDX- 110 material was obtained at I 0 rng/mL in 10 mM PBS pH 74 5 Buffer exchanges of the CDXI 10 material into the above identified formulation solutions were carried out with Amicon Ultra15 MWCO 50 centrifugal concentration on a Beckman Coulter Allegra 21R centrifuge run at 4500 RPM at SC. Approximately eight volume exchanges were made and the solution concentrated between 1.4 to 1.8 mg/mL. Approximately 70 to 99 mL of all formulations were prepared. CDX-110 concentration 10 was determined by Ultraviolet-Visible spectrometry (U-Vis} method using an extinction coefficient of 1.38 (mg/mL cm at 280nm. .A 400 mg/mL disaccharide solution was prepared by dilution and dissolution of tiehalose/sucrose by the appropriate formulation buffer as described in Tabie IA. In addition, a 10 mg/mL polysorbate 80 (PS80) solution was prepared by dissoluon of 15 PS80 in the appropriate formulation buffer Appropriate volumes of the disacchraride stock and PS80 stock solution were then added to the CDX-110 stock solution to obtain a final concentration of 1.0 mg/mL in the formulatiori compositions listed in Table IA. The formulations were then filtered through 0.2 pm sterilizing filters and filled into vials.. A fill volume of 0.6 mL was filled in 2 mL type I glass vials. Liquid formulation 20 vias were stoppered with Daikyo 71 Flurotec@ coated serum stoppers crimped and sealed Lyophilized formulation v~is were partially stoppered with Daikyo 777-I Fiurotec@ B2-TR coated, then lyophilized according to the freeze drying cycle parameters outlined in Table 1D, stoppered, crimped and sealed, Both liquid and .yophilized formulations were placed in stability chambers stored at , 25 and 40Qc for 2, 25 45 8, 13, 26, 52 and 104 weeks. The vials were washed and autoclaved as were the 13 mm Daikyo 777-1 serum and lyc stoppers The lyo stoppers were dried at 100* for 8 hours. The samples were analyzed by appearance, pH, and protein concentration by UV-Vis absorbance, size exclusion HPLC, Anion Exclhange HPLC, Reverse Phase HPLG. The. study is ongoing and values reported are from intermediate time points or 30 12 weeks.

34 Table 1D: Freeze Drying Cycle Parameters ________StP TemperaturelPressu re Freezing 400 Annealir -21 OC Freezn 40*C Primay Dr-25'C CaMber u 100 rt)T Secondary na4 cw tolig_ _ _ _ _ _ _Urdernitro~en Assays Appearance 5 Each formulation was visually evaluated after 0 initial) , and defined time points for particulate formation, color change and clarity, The sample was held against a light and dark background with proper iuumination, The sample cannot be more intensely colored than level 6 of the color range and not more opalescent than refrerice suspension IlIl Visible, particulates were also reported. 10 pH Samples were subjected to a potentiometic pH measurement pH meter calibration was done using commercial standard buffers ranging from pH 4 to 10, Protein Concentration by UV-Vis Absorbance Protein concentration measurement was carried out using a suitable 15 spectrophotometer, The sample as scanned at 280 nm and the absorbance at 280 nm was used to determine the concentration of the protein, An experimentally derived extinction coefficient of 19 (mgnm cm and 138 (mg/mL cn< (for D liq and D lyo) at 280 nrn was used to determine the protein concentration. Size Exclusion HPLC 20 The presence of monomers, dimers and high molecular mass species (HMMS) is monitored using size exclusion chromatography (SF04 The size exclusion chromatography was carried out using Waters Biosuite 450 SEC column, mobile phase 50 mM sodium phosphate, 75 mM NaCi, 0,01% sodium azide, pH 74, a flow rate of 03 rmt/min. and UV detection at 214 no. The levels of monomers, dimers and HMMS were 25 calculated by integrating the area under the chromatogramn peaks for each formulation and reporting the integrated areas of monomer dimer and high molecular mass species peaks as a percentage of total peak area.

-3s Anion Exchange HPLC The anion exchange chromatography was cared out using TSKgei DEAE-5PW column, with mobile phase 10 mM Tris, pH 7.3, and 10 mM Tris, 0,5M NaCl, pH 73 5 gradient elution, a flow rate of 1 mUmin. and UV detection at 280 nrm. Anion exchange chromatography separates CDX< 10 from KLH, peptide, inker and related compounds based on their partitioning between a charged stationary phase and a mobile phase of varying ionic strength. The species of interest are detected by ulaviolet absorbance and quantitated by relative area. 10 Reverse Phase HPLC Reverse phase chromatography was carried out using Waters symmetry 300 C4 column. with mobile phase 0.1% TFA water, and 0.1% TFA in acetronitrile gradient elution, a flow rate of I nUrmin, and UV detection at 215 nm Reversed phase chromatography separates CDX- 110 from peptide and linke-related impurities on their 15 partitioning between a nonpolar stationary phase and a polar mobile phase. Peaks are detected using an ultraviolet light detector at a fixed wavelength '. where the peptide and Ink er based impurities have absorption Results Appearance 20 From the appearance analysis, it could be concluded that ali formulations in sodium phosphate (B), potassium phosphate (A, C & D) and histidine (E) behaved similarly in both the liquid and lyophiiized formulations. Formulation J (liq, Succinate) and F (lyo, PBS) behaved the worst, For details, see liquid and lyophilized formulations outlined in Tables IA, 1 , and IC. 25 Liquid formu/aton (Table 1$ All TO (time zero) formulations were reported colorless and NMO RSI. in terms of articulates, I white flake particle was observed for formulation D (liq KPi, Te) whereas formulation C (lig, KPi, Tre), which only differs in trehalose content by 6 mg/mL, is EFVP essentially free of visible particles (EFVP) Current formulation F (liq, PBS is also 30 colorless and EFVP -as 3 At 5*C (T=12 weeks), all formulations were colorless and NMO RSIL 1 particle was observed for the B (liq) and C (liq) formulation whereas current formulation F (in PBS) remained EFVP and NMO RSt At 25"C (T= 12weeks) J (Rik succinate - NMO RSII) and D (lig KPI, Tre NMO 5 RSI) formulations were reported to be very slightly brown. F (1 q, PBS) formulation was reported to have a number of different types of particles while D (liq, KR, Tre) had 1 particle. A: 4 0"C (T=12 weeks) all formations changed color to yellow or brown (except PBS Le, F) with J (liq, succinate) being the worst in terms of opalescence (NMO RS V), 10 Potassium phosphate liquid formulations C (liq, KPi Tre) and D (liq, KPi, Tre) were reported to be very slightly brown with one or no particles Fornulation F (1q PBS) was colorless with two white flake-like particles. Formulation F (lhg, PBS) formulation was the only one without PS 80. Lyophiized formulations (Table 1E) 15 All formulations at T=O were colorless except for F (lyo, PBS) formulation (slightly brown on reconstitution) which also had many brown/tan particles resembling cardboard packaging. A (lye, KPi, Suc) also had one particle on reconstitution, At 5"C (T=12 weeks) all formulations were colorless and EFVP except for F (lvo, PBS) which had many particles and opalescent. At 25"C (T=12 weeks) all formulations 20 except F (lyo, PBS) were colorless and NMO RSL Two small white particles were also reported for C (lyo, KP Tre) At 40*C (T=12 weeks), ali formulations were colon'ess and NMO RSI except for F (1yo, PBS) on reconstitution One and two particles were observed in B (lyo, NaPi, Suc) and D (lyo. KR, Tre), respectively, while F (lye, PBS) had multiple particles. 25 Concentration Liquid fomnu/ations (Table IF) At 5*C (T=12 weeks) no significant change was observed. At 25C (T=12weeks), an increase in concentration was observed for E (q His, Tre) fornulatlon All other formulations did not show any significant change At 40*, formulations C (lig, KPi, 30 Tre), D (lip, KP, Tre) and F (lig PBS) did not show any significant change while A (lig; KPi, Suc), B (iq, NaR. Suc) and E (liq, His, Tre) showed a marked increase in concentration.

L yophihzcd fonulations (Tabel1F) At 5C (T=12 weeks) no significant changes were observed except for F (lyo, PBS) which showed a slight drop in concentration At 25"C (T=12 weeks), o signficant changes except for a slight decrease for E (ly, HisT Tre) and F (lyo, PBS) At 40tC 5 (T=12weeks) no significant changes except for F (lyo, PBS), A slight increase was observed for A (lyo, KPi, Suc) A280/A340 Rafio The ratio of absorbance at 280 nm to absorbance at 340 assumes importance due to the presence of bound copper in CDX-10 which gives it a characteristic blue 10 color. The 340 nni absorbance is due to the presence of copper and therefore a reduction of absorbance at this wavelength indicates a ioss of copper or change in copper oxidation state. Liquidformulations (Tale IG) At 5*C (T=12 weeks) there were no significant changes in the ratio except for 15 formulation A (lip, KPi, Suc) where an increase is observed. At 25'C (T=12 weeks) al liquid formulations show an increase in the ratio indicating loss/changes to the copper molecule except for formulation J (iq, succinate). Formulation J (lig, succinate) showed a decrease in the ratio unlike the other liquid formulations. Some of the formulations showed a greater change in ratio compared to others eg formulations C (lip, K~l, Tre) 20 D (li, KP, Tre) and F (lip, PBS) had a more significant increase than A (1iqK Sue) and B (iq, NaPi Suc) E (4iq, His, Ire) had a moderate increase in the ratio. At 40*C, formulation J (lig, succinate) shows a significant decrease in the ratio (Table IB). Formulations C, D and E show a significant increase with F showing the highest increase, The ratio for sucrose containing formulations A (liq, KPi Suc) and B 25 (14 NaPi. Suc) appear to be constant, A closer look at the data revealed that the sucrose containing formulations show an increase in absorbance at 280 nrn as well as an increase in the absorbance at 340 nm with time and temperature, thus allowing the A280 to A340 ratio to appear to be constant. For trehalose and PBS containing formulations, an increase in this ratio is observed due to a decrease in the absorbance 30 at 340 nrm, L.yophilized fomwlations (Table 1G) At 5C (T=12 weeks) no/minimal change observed except for F (Iyo, PBS), At 25CC no/hninimal change observed except for F (lyo, PBS) At 40C, no significant change in the ratio is observed for all formulations except for F (yo, PBS). 5 Dimer/MonomerHMMS Liquid fonnu/ations (Tables 1K 1 and 1J) Dimer content (Table 1H) decreased for all formulations with increasing temperature. The smallest decrease was observed for C (lig KP. Tre) and D (1p KR, Tre) formulations. Monomer content increased for all sugar containing formulations 10 whereas the histidine (E) and PBS (F) formulations showed a decrease. The histidine formulation showed a marked decrease in mononer content (Table 1 H) at 25 and 40C with a simultaneous increase in the HMMS content (Table 1 ). An exact opposite trend was observed for the sodium phosphate, potassium phosphate formulations where an increased formation of monomer from HMMS occurred at higher temperatures. 15 Lyophilized formu/ations (Table 1ffH I Iand 1J) All lvophlized formulations showed no/minimalchanges at 51 25 and 40*C at the end of 12 weeks except for the lyophilized PBS formulation (F lyo, PBS). Percent Impurity and COX1 10 retention time changes by AEK Liquid formnulators (Table Ki and IL) 20 At 54C (T=12 weeks) percent impurity for all formulations was less than or equal to 1% except for the succinate formulation 0), Retention time for the CDX-110 peak moved approximately one minute for all formulations except succinate The shift in retention time indicated formation of acidic species which can be attributed to a combination of linker hydrolysis and deamidation At 25 C and 40*C (T=12 weeks) 25 percent impurity increased significantly for all formulations. Change in retention time also followed a similar trend indicating increased formation of acidic species. Lyohi/zed formulations (Table 1K and IL) For all lyophilWed formulations the percent impurity change and the shift in retention times at all temperatures were not significant except for the F (iyo, PBS) 30 formulation.

39 -9 Percent Impurity by RPHPLC lquid foruaon (abe 1M An increased formation of peptide and linker related impurities were observed for formulations with increasing temperature, with histidine formulation (E) showing the 5 least formation of impurities, Lyophi/lzed fbnulations (Table 1M1) No significant change was observed for any of the formulations except for the 1yophilized PBS (F) formulation, Mouse potency 10 Drug product specification for potency is set at :75% seroconversion in mice. Formuiations tested were potassium phosphate (A, C D) histidine (E) and PBS (F)P Samples tested were stored at 50C for 8 and 12 weeks. Ali samples demonstrated >75% seroconversion (Table IK) and therefore met the drug product specifications, 15 Table 1E: Appearance Data for pHlbuffer Study Formulation Appearance Appearance at Appearance at 25C Appearance-at at T=O wk 5C T=i2 wk T-12 wk 40C Th, 2wk N'sVO RSI NMO RSI NMO RS 'N 8 IV C okiress dolores 89)~ V I brown (<57) Srown (<BS) sutcinate PS80 B EFVP EFVP 2 flc (white pH 4, [,3,q EF VPgobuar tNVO PiS N& NiMO SS A q) Coloriess Colrle~ss 89) oloress (<9 Si brown yehow H 9 EFVP EFVP (<BY6) EFVP Ismaiwhite _____________ __________ ____________ ______________ round particle NMO0 RSI NMCO Rf NM) RS NM RS! A lyochess Icktiesb9 Colarkos (<B9) Cooress (<89) KPO. Sun'0 I(-< VP EFVP EF mginM PS80 p'H I short white 7.4 yo erie NM W 0 RS5 NI) RS I N-MO RS NM C R SI NaPO %c 0 CoVkt3Cooes <9) (Coloriess (<B39) Si Brhown YeV ow mg/m P80 pH (<9 1 wEF VP EFVP Fs iberVke EFVP NaPO S-840 CokyrIes Coors (<29) Cloriess B 9) Cooriess (<9' mg/ml. PS80, PH IB) EFVP EFVP short whit 74. 4o EFVP "beike ite C (ig NMO R,),] NMOR N-MOPS! NMO RS KPO T e4a4 Colri-s Colorlesc &c9) C okess 89) Si brown yelow m/O P OoH i I 1shrwhws [FVP .. ...............

40 Formulation Appearance iAppearance ; -at Appearance at 26C Appeare at )t=wk SCT=1 lwk T=12 wk4O hlw a~e an w' and 3rima wh e found parties C yo) NMO RS I N M0 RE NM) RS NMPO RS KP04, re F4 Coorrkss Cokless (439) olorless (89i) solorles (<9) mg/ml PS80, pH (fB9) EFVP 2 small white round FVP 4S n EFVP artide N MR PSI NMO RSi NMD RS NMQ RSi Kp&4. s Co-oriess c <r ( 89) v siht brown (<7) v &ght brown m B9 EFVP 1ea whtound (<87) IA. ~ 1 wciite flake particle EF P 7A, paricl ul' N R N &Opt S N V;0 PS NMC fv 0RJ VPO4 Tre 9 Coler es Cozhless (' B9) corioss Bc8) Colorless (<$9) mgn-m PS0 pH vB9' FVP EFVP 2 smnall whte k-tq V)P- hNMO RSN{IPINDRIS I -- ' 4 Colasrss Colsas ( 19) Coloriess B8) Brown Yellow mg/ml PS5G p-H (<B9) EFVP E FVP (<36) EFVP E F VP E (lye) NMC I NMO R PIS N S NM RS His Tre-84 Coloriess Co orles 89) Colorless (<89) Corloess (<OB mqr~. 5' vOp -pvp mhlPS80,PH (<B9) EFV EFVPEV f'.5. !ye-F -v NMO~ R$ N/O R-t NMO R 0l N 5TC ColeIessas 89) Coloress ( B9) - CoBssg (<19) (<9EFVP hort white fr like 2whte ftke ke F EFq IIVP partilers 5 maN partces PBS pH 7 .q white glouar pa t--les ~- - sma white rake like NN) P-I NMO PHI N\MO) RP3 NMVO R311 S-4igtiy brown Very bSIighv VEW $1lhlylVBrownl Very Sliignly (<Bt) Many Brown (<P -10 (-B7) 2 short white Srown (587) 3 5 short tan short fierke fiberlike and 4 white tan short Fbtrown tn n -- wJne globulr parties ibere, 2 small fears. Typica gobulr particles white round 5 f 7.4 ardbeard oc white lobular pa'c-kagi. g partic fOca sho-t white s---verlike ___ partes ____ _ ___ ___ EFVP: Essentialiy free of visible part-ees; NMO RS MUMli Not more op alescent than reference suspension 11111 Formulation J was disconinued after 8 weeks Data reported is at the end of 8 weeks 5 Table IF: Change in Concentration Fomlulation Change in Change in Change in concentration concentration concentration (mn/mL at 5C in (mgnL) at 25C in (mg/mL) at 40C in 12 wks 12 wks 12 wks Sucinate P5e pH -005 001 0.40 4.5 Iiq" I I I -4) Fomaulation Change in Change in Change in concentration concentraion concentration (mg/m4) at 5C in (mgim ) at 250 in (mg/mt) at 400 in 12 wks 12 wks 2wks A (P4 KPOQ So-8o mgm 0 14 0,04 0 35 A ft KPOI: Suc mg m 008 03 3 gmPS80, PH 74 m'c h 11 PS l0, 0__ _ _ _ _ Ell, -0, C, 0, 0 8 0 7A NaPO Suc-8o gmqsm. p~QH 7A 404 0.05 03 KPO04 Tre84 mg 00003 .107 Clyo KPO4 T 4 mg/m 0 03 0 02 0.09 PS80 pH hy 4oq D k KPO-4 Trea-90 m, 002 0. 0 402 D80,pH 7A yo KPO4, Tre40 mg n 0 02 0 A4 8 PS 4 mghn 0.05 0(18 0 4 Heo, Tre 04 mg0, 07 0.1 0 P H4 q 02 0.03 0.05 F (tyo) FormulanJwaor s dpa cont wed after 8 weks. Dat reported s at the end of 8 weeks Table IG Change in A280/A340 Ratio Formulation Change in Change in Change in A280/A340 at 5C in A280/A340 at 25C A280/A340 at 40C 12 wks in 12 wks in 12 wks Su'ccinae. P80. pH 0 -054 -4 12 A 4) KPO, Suo 80 m 0 0,45 P380 pH74, 9 A Ovo)

KP

4 Sucn 80 ng/mnt 402 001 0 12 PS60, pH 7.4, lyo 42 Formiilation Change in Change in Change in A280/A340 at SC in A28WA340 at 25C A28WA340 at 40C 12 wks in 12 wks n 12 wks NaPO S 8 0421 0 A5 0,21 a yo NAPO Su 80 -0,02 0,06 0,06 mgi mO Ps300, pH 7.4 KPO4 Tre 8 mgm 0, 01 2 79 5 5 Ps80r pH_'4_S q _________ KPO Te84 rng/m 0.09 0,03 0.98 D (iq K-O4 Tre-90 mgrn: -0.01 259 5.45 DS8,$, pI 74 9q KPO4 Tre-0 mg/nt. m.13 0 0.20 P0. pH 7.4 yo E " Iiq Hk: Tre 84 mg/mb 0.01 53 3,98 E fl& H s,'fra44 mgm, 0.02 0.24 0.20 0.02 2,59 95 rF 0,34 0,03 098 Formuator J was discontinued after B weeks Data reported s at the end of 8 weeks. Table 1H Change in Percent Dimer Forilation Change in dimer % Change in dimer Change in dirnr at 5Cin 12 wks % at 25C in 12 wks % at 40C in 12 Sucinate, PS80 pH NM NM NM

KP

4 Suc-0 mg 0,12 4,08 -41.98 11 H 7 g, q A >o)7 KPO Su. 80 mg/m 1 50 150 0.70 0 mq) NaPo Sun-0 0 00~H~ 0.13 4.93 .45.73 g fra, PS80, PH 74, ..................................... NaPO SunSO mg/mi PB0, pfH 7: 20 4,A0 -0.8 Iye -43 Formulation Change in dimer % Change in dimer Change in dimer at SC in 12 wks at 25C in 12 wks % at 40C in 12 wks KPCO4 84 mg/0.19 441 -14.61 KP04 Tre-84 mg/V 1.70 1.10 -0,30 PS80 pH 7 4 Igq KPO4, fre90 mngrN, 0,90 0 - 8 PS60 ~ y H is ', 84 mgm-0.29 -341 -46.71 E y 4 .re4 060 030 q) 45 -2Z35 -2,35 Fas, a 10 3080 Formiulation J was dilsconfinted after 8 wekeData report, at tkhe end of 8 weeks: NMnot measured since ass:,ay miethods were different when the studIy was performe-d, Table it. Change in Percent Monorner Formulation Change in Change in Change in rnonomer % at 5C monomer % at 25C mnromer %k at in 12 wks in 12 wks 40C in 12 wks J VS dae PS80 pH NM NM N A iq) KPO, Sue : 80mgm2 4.64 26.94 pA l q , s2 0sm0 0, 70 -020 NaPO't Su5 9252 35 gmPS8 pH7 4q... . . -030 5.20 -00 gm PS0 pCH 4 it iq&

KPO

4 . T-4 mgm. 0.2 424 8 30 KPO4, Tr-e44 mg/mn ~0 80 -060

KPO

4 0 mg/m 7,1 4.0 0 C11.30 - 44 Formulation Change in Change in Change in monomer % at SC monorner % at 25C mnonmer % at in 12 wks in 12 wks 40C in 12 wks iK e 00 mg1 010 0,00 0.30 E { iqg H s T,4 0.94 2.96 -19.34 E y HZ 7 mglmL -0,40 -020 -0.40 PS 80q pH 6. Nyo 0,78 2 28 -3.02 PBS pH 4 q 080 -2.00 2.70 Formuhation J wasiscontinued after 8 weeks. Data reported is at the end of 8 weeks; NM-not measured since assay methods were different when the study was performed, Table iJk Change in Percent HMMS Formulation Changein Changein HMMSSChange in HMMS % at SC in 12 wks % at 2SC in 2 wks % at 40C in 12 wks Sucinate Ps0, PH NM NM NM KP0 4 sue aomgh, -0.33 263 -12 53 A Oyo) KPO( 'in'80 mnm -0-30 0.50 040 V-RO 6"PH 7.4. lye NP Su 7-1,27 -297 -12.97 mrrtPS80, PH 7.4, VPO Su1N mg/mO PS30 pH 74 Iyo KP4, e84mm 6 -2,36 -6-66 PsaM pH74. Hq KP4 Te 84 mg -06 0.40 0,60 PS80 pH 7.4, !yo ____________________ :3 hq) KPA I 9r- megh 010 1.70 -4,30 D y 4 20 KPO4 Tre- 90 mg m 0 21 _ _ _-__ _ _ _ : -_ _ _ 2-__-_ E___ -0802,20 M40 -45 Formulation Change in HMMS Change in HMMS Change i HMMS % at 5C in 12 wks % at 25C in 12 wks % at 40C in 12 wks H1± TH (4 mgq E 0. ' Tre 54 mg'm' 010 '30 0.90 F PBls, pH T4 hq F (PyN Frmutldon J 'wasOoninued after 8 weeks Data epor td ae endo 8. weeks Nr measured scea methods were different when h e study wasprfr Table 1K: Change in Percent Impurities by AEX Formulation Change in AEX % Chanig in AEX % Change in AEX % inpurities at 5C in impurties at 25C impurities at 40C 12 wks in 12 wks in 12 wks Succnate, PS80 pH 3,45 5.80 1770 'tig KOSu 80 mg/mh 0.30 .20 1 60 KPOn pH 4,Bc A iO~ KPO Sue 80 mg. 0.20 0,20 010 NtaPOi Suc43{ nH 7.4, 0.10 1 .00 1,30 B Oy 'o NaPO Suc80 0,10 0,20 0,10 KP4 84 mg/lt 010 0,9 2,60 P380 pH 7A ig KP04 re 84 m/rlgh 0 20 0,10 KPO4, T:re9 fmgm 1.00 2,70 350 PS80, pH 7 4 y E . g) V sF"s mg mqr -0.80 2370 6.70 P380 pH65 g His Tr-4 mgsn, -1.40 120 -0,90 FIBS, pH 7 4 0q 0.0 330 0',~T 4 lyeZ 30 -46 Formulation J. was discontinued after 8 weeks, Data reported is at lhe end of 8 weeks. Table I L: Change in AEX Retention Time Formulation Change in AEX RT Change in AEX RT Change in AEX RT (mins) at 5C in 12 (minsO at 250 in 12 (mins) at 40C in w Wks 12 wks Sucdnate PS80. PH 1,21 3,02 A l g KPO4 sut a1 m 1.05 2,93 8,91 PS80 pH_74_P ____ig____ A 0 KPO4 Suoc40 mg/m 0.80 0,99 0,97 ' w 4, yo Biq~ t~aPO ScS Py RSu 80 0,93 2,97 8,65 NP gr PS30 oH 7.4. N lP Se 80______ KPO re 4 mg/mt 0.84 2,0 6,27 KP04 Tre-84 mgm 074 0.90 0,81 P58 10. pf-i17,4 lye o ___________________ KPO.4 Tre-C ngh 0.76 .98 5.28 PS80 pH 7A le KP04, Tr/90 uim, -0,04425 Hts Tr4 mg/mi. 0,93 2,19 4,98 P380 PH 6,5, 0i9 _ _ _ __ _ _ _ _ __ _ _ _ _ _ H~ I e n4 mgm 0.80 0.90 083 F ii)1,05 35 6M9 F Ovo 0.98 60 2.8 *ormulatio r J was discontinued after 8 weeks .Dat reported is at the end of 8 weeks 5 Table IM: Change in Percent Impurities by Reverse Phase Formulation Change in RP Change in RP Change in RP impurities % at 5C rnpurities % at impurities % at in12 wks 25C in 12 wks 40C in_12wks Succinate PS80. pH NM NM NM - 47 Formulation Change in RP Change in RP Chmnge in RP inpurities % at 50 impurities % at impurities % at in 12 wks 25C m 12 wks 40C in12 wks A (RO KPO, S8 mg m 1,90 3,10 11 00 A yo KPOI Suc8 mg/m 1 10 0 90 0 90 B (gy) NaPO Su&80 mqsm,2 0s3 0,J4A 3,40 14M8 gq s'o 7 0, 90 0.70 0 70 C (q KPO4 Tre-84 mg/n 90 3.0 10.00 Pz.,3, p8,I 7.4, 9iq ___________ _________ C Oyvo) KPO4 Tre-4 mgm' 1 0 1.10 0.80 PISK0 H7Ayo KPO4 T e90 mg m 0,40 1.90 8.00 KPO4 Tre-90 mg/m -010 ~020 440 PS&'13 p11I 74, lye __ _ _ _ _ _ _ __ _ _ _ _ _ _ _ Sao mg/ 2,0 3,0 3.10 P0pHG i RH 6,5, 5q Te 84 mgm 0 90 0.80 0.80 -P 40 pH 6.5 lye 1.50 3'30 5.70 PBS, pH 7,4, hq F typ)f' 0F 00 0.10 2.70 WftB ILS f ~ I d Formnadon J w~vasscontinued after 8 weeks Data reported is t the end of 8 weeks NM-not measured since assay methods were different when the study was performed, Table IN Mouse Potency Results Formulation % Seroconversion* T=0 T 8 weeks T 012 weeks A (lyo) 83 NM NM C (fiq) 100 NM 100 C (lyo) 100 NM 100 D (liq) 92 NM NM D (1yo) 100 100 NM -45 Formulation % Seroconversion = I -- weeks T012 weeks F q 100 NM G(iq) 100 NM NM E (q) 100 NM NM E (lyo) 100 NM NM DP Spec 75%; NM: Not measured. Example 2: Effect of Disaccharides A study was carried out in which various liquid and Iyophibzed formuations were 5 prepared to study the effect of disaccharides at pH 7.4. Trehalose and sucrose were used for the study. The C and D formuations have two different concentrations of trehalose with the rest of the components the same. The protein concentration and pH was kept constant as shown in Table 2A. Potassium phosphate and sodium phosphate suffering species were used for these -formulations A comparisons are made against 10 the current formulation (formulation F both liquid and lyo) in PBS (see Tabie I B), Table 2k Formulation Compositions for Effect of Disaccharide Fornuation A A B B C C D D F F (ihq) (yO) (liq) (lyO) iq) (tyO) (liq) (lye) (tiq) (lO) CDXl10 i 10 1.0 1.0 10 1.0 1 0 0 t 1.0 10 mg/mt { Na 10 10 Phosphate mM K Phosphate 10 0 1 10 10 mM Histidina mM Succinate mM 7 4 4 774 74 7 4 74 7A 74 Sucrose, 80 60 80 80 mgim.I Trehalose 84 84 90 00 mg/mtj Tween8O 0.2 A2 .2 02 02 12 02 02 mg/n -4 v=IO-6 0.6 d dOW CUK I Uid 0 06 PBS composition as given n Table 1I Formuaions C and D differ only n trehalose content by 6 mgmL. Ai lyopilized formulations were reconstituted with WFI suh hat final CDX-110 concentration was -1, mg/mL. The analytical assays performed for stability were Appearan'e IUV pH. SEC, 5 AEX and RPHLC. Tables 2B - 2K show data on stability for multiple degradation pathways induding change in monomer/dimerhigh molecular weight species content, formation of peptide-linker related impurities, change in A280/A340 ratio, AEX retention time of the CDX-110 peak due to formation of acidic species and mouse potency studies, 10 Preparation of Buffer Solitions Five buffer solutions were prepared as described in Table 2B below. Each solution was prepared by first dissolving an amount of the buffer species (listed in Table 2B) in water for injection (WFI). The pH of each buffer solution was measured. The buffer solution was then filtered through a sterlization filter (022 micron pore size) irto a 15 sterilized receptacle for subsequent use, Table 2B: Buffer Solutions Buffer Type pH Buffer Species Butfer Strength (mm) Histidine 6,5 LHistidine 15 L-Histidine HCI monohyd rate Succinate 4 5 Suc nic acid 10 SIum Succinate PBS 74 Sodium phosphate dibasc 10 anhydrous (phosphate bufe Potassum phosphate monobasic Saline) Sodium chloride Potassium chloride potassium Phosphate 74 Potassium phosphate dibasic 10 anhydrous Potassium phosphate monobasic Sodium Phosphate A Sodin Phosphate. Monobasic 10 mooydrate Sodium Phosphate. Dibasic, Anhydrous Preparation of CDX410 Formulations -50 The formulations that were evaluated are listed in Table 2A, To prepare each formulation. CDX-110 material was obtained at 1.0 mg/mL in 10 mM PBS pH 7.4. Buffer exchanges of the CDX-1 10 material into the above identified formulation solutions were carried out with Anicon Ultra15 MWCO 50 centrifugal concentration on a 5 Beckman Coulter Allegra 21 R centrifuge run at 4500 RPM at 5-CS Approximately eight volume exchanges were made and the solution concentrated between 1.4 to I8 ng/L Approximately 70 to 99 rt of all formulations were prepared CDX-1 10 concentration was determined by Ultraviolet-Visible spectrometry (U-Vis) methods using an extinction coefficient of 1.38 (mglnL< cm at 280nm, 10 A 400 mg/mL disacchadde solution was prepared by dilution and dissolution of rehalose/sucrose by the appropriate formulation butteras described in Table 2A. In addition, a 10 mg/mL polysorbate 80 (PS80) solution was prepared by dissolution of PS80 in the appropriate formulation buffer as described above. Appropriate volumes of the disachraride stock and P80 stock solution were then added to the CDX-1 10 stock 15 solution to obtain a final concentration of 1,0 mg/mL in the formulation compositions listed in Table 2A The formulations were then fitered through 0,2 pm sterilizing filters and filled into vials. A fil volurne of 0, rL was filled in 2 mL type I glass vials. Liquid formulation vials were stoppered with Daikyo 777-1 Furotec@ coated serum stoppers crimped and 20 sealed, Lyophilized formulation vials were partially stoppered with Daikyo 777-1 FlurotecE B2-TR coated, then lyophilized according to the freeze drying cycle parameters outlined in Table 2C stoppered, crimped and sealed. Both liquid and lyophilized formulations were placed in stability chambers stored at 5, 25 and 40*C fcr 2, 4. 8, 13 26, 52 and 104 weeks The vials were washed and autoclaved, as were the 13 25 mm Daikyo 777-1 serum andlyo stoppers The lyo stoppers were dried at 100"C for six hours, The sarnples were analyzed by appearance pH, protein concentration by UV Vis absorbance. Size Exclusion HPLC, Anion Exchange HPLC, and Reverse Phase HPLC. The study is ongoing and values reported are from intermediate time points or 12 weeks. 30 Table 2C Freeze Drying Cycle Parameters Step TemplPressure Freezing -40c Annealing 21 0 Freezing ~401Y Primary Drying 25 Q Chamber Pressure 100mT Secondary Drying 40YC Stoppering Under nitrogen Assays Appearance Eacd formulation was visually evaluated after 0 initial ) and defined time points 5 for particulate formation, color change and clarity. The sample was held against a light and dark background with proper Illumination, The sample cannot be more intensely colored than level 6 of the color range and not more opalescent than reference suspension IllL Visible particulates were also reported. pH 10 Samples were subjected to a potentiometic pH measurement. pH meter calibration was done using commercial standard buffers ranging front pH 4 to 10, Protein Concentration by U V-is Absorbance Protein concentration measurement was carried out using a suitable spectrophotometer, The sample was scanned at 280 nm and the absorbance at 280 nm 15 was used to determine the concentration of the protein. An experimentally derived extinction coefficient of 1,19 (mg/mL" cm arid 138 (mg/mIL)" r mfor D iq and D lyo) at 280 nm was used to determine the protein concentration. Size Exclusion HPLC The presence of monomers, diners and high molecular mass species (HMMS) is 20 monitored using size exclusion chromatography (SEC). The size exclusion chromatography was carried out using Waters Biosuite 450 SEC column, mobile phase 50 mM sodium phosphate, 75 mM NaCi 0.01% sodium azide, pH 7.4 a flow rate of 03 mULmin, and UV detection at 214 nm. The levels of monomers, dimters and HMMS were calculated by integrating the. area under the chromatogram peaks for each formulation 25 and reporting the integrated areas of monomer, dimer and high molecular mass species peaks as a percentage of total peak area. Anion Exchange HPLC The anion exchange chromatography was carried out using a TSKge DEAE 5PW column, with mobile phase 10 mM Tris pH 7,3, and 10 mM Tris, 0.5 M NaCL, pH - 52 7,3 gradient elutior. a flow rate of I mLimin, and UV detection at 280 nm, Anion exchange chromatography separates CDX-1I10 from KLH, peptide, linker and related compounds based on their partitioning between a charged stationary phase and a notbie phase of varying ionic strength. The species of interest are detected by 5 ultraviolet absorbance and quantitated by relative area. Reverse Phase HPLC Reverse phase chromatography was carried out using a Wates symmetry 300 C4 column, with mobile phase 0 1% TFA water, and 0 1% TFA in acetronitrile gradient elution, a flow rate of 1 niUmin, and UV detection at 215 nm. Reversed phase 10 chromatography separates CDX-110 from peptide and inkerrelated impurities on their paritioning between a norpolar stationary phase and a polar mobile phase, Peaks are detected using an ultraviolet light detector at a fixed wavelength where the peptide and linker based impurities have absorption. 15 Results Appearance From appearance analysis, it could be concluded that sucrose and trehalose formulations behaved similarly in both the liquid and lyophilized formulations. Formulation F (lyo, PBS) behaved the worst. Further details are noted below. 20 Liquid formulations (Table 2D) All TO formulations were reported to be colorless, In terms of particulates, I white flake particle was observed for formulation D (li, KPi, Tre) whereas formulation C (lrq KPi Tre), which essentially only differs in trehalose content by 6 mg/mL is EFVP Current formulation F (lig PBS) is also colorless and EFVP, At 5QC (T=12 weeks), al 25 formulations were colorless and NMO RSL One particle was observed for the B (lig, NaPi, Suc) and C (lip KPi, Tre) formulations whereas formulation F lip PBS) remained EFVP. At 25*C (T=12 weeks), for-mulation D (iq KP, Tre NMO RSI) was reported to be very slightly brown. Formilation F (lig, PBS) was reported to have a number of 30 different types of particles while D (lip, KPi, Tre) had one particle. At 40"C (T=12 weeks). afl formulations changed color to yellow or brown except formulation F (lipg PBS). The trehalose formulations C and D were reported to be very slightly brown with - 53 one or no particles, Formulation F (lig, PBS) was coloress with two white flake-like particles, Formulation F (i4 PBS) was the only formualtion without PS 80, Lyophiized fonmulations (Table 2) All formulations at T=O were colorless. A ciyo. K.i Suc) was also reported to 5 have one particle, At 5C (T=12 weeks) a formulations were colorless and EFVP. At 25C (T=12 weeks) all formtulations were colorless and NMO RSI Two small white particIes wore reported for C (lyo, KPi, Tr) At 40&C (T=12 weeks), all formulations were colorless and NMO RSIL One and two particles were observed in B (lyo, NaiR. Suc) and D (lyo, KPi, Tre) respectively. 10 Concentration Liquid formulations (Table 2E ) At 5 C (T=12 weeks) no significant change was observed, At 25 C (T=12 weeks); all formulations did not show any significant change. At 40C, formulations C (liq, KPi, Tre) D (liq KPi Tre) and F (iq PBS) did not show any significant change 15 while A (jiq, KR, Suc) and B (lig. NaPi Suc) showed a marked increase in concentration. This demonstrated that th e trehalose formation behaved better than the sucrose formulation. Lyopbilized formulations (T able 2E): At 5"C (T=12 weeks) no significant changes were observed, At 25"C (T=12 20 weeks) no significant changes except. for formulation F (lyo, PBS). At 4O0 (T=12 weeks) no significant changes except for formulation F (lyo, PBS), A. slight increase was observed for A (Iyo, KP Suc). A 280/AA340 Ratio Liquid fomiaulaons (Table 2F) 25 At 5C (T=12 weeks) there are no significant changes in the ratio except for sucrose formulations (A and B) where an increase is observed, At 25 C (T=12 weeks) all liquid formulations show an increase in the ratio indicating Loss/changes to the copper molecule Some of the formulations showed a greater change in ratio compared to others e.g. formulations C (lip, KPi, Tre D (liq, KP Tre) and F (liq, PBS) had a more 30 significant effect than A (lipg KR, Suc) and B (iq, NaPi, Tre), At 40C, the ratio of the suCrose containing formulations A (lig, KPi, Suc) and B (lir, NaPi, Suc) appear to be least affected (Table 2F) than the other formulations. The concentration data (Table 2E) shows that formulations A (liq, KPI, Suc) and 8 (1iq NaPi, Suc) had significantly high concentration (or high absorption values at 280 nim values at 40*C In addition the 5 absorbance at 340 nm also increases with time for the sucrose containing formulations, thus resulting in little change in the A2801A340 ratio. For the trehalose containing formulations C (li, KPi, Tre), D (iq, KR, Tre) and the current PBS formation F (1ig, PBS). this phenomenon (Le, no increase in A280 with tinme) is not observed, leading to an increase in the A280/A340 ratio, largely due to the decrease in absorbance at 340 10 nm, Lyoephiized formulations (Tabl 2F) No significant changes were observed in any of the lyophilized formulations except for formulation F (lye, PBS). DinerMonomer/HMMS Content 15 Liquid tormouaulons (Table 2G, 2H, and 21) Dimer content (Table 2G decreases for all formulations with increasing temperature. The smallest decrease was observed for the trebalose containing formulations C (liq KPi, Tre) and D (liq, KR Tre), Monomer content increased for all disaccharide containing formulations whereas the current formulation F (lig, PBS) 20 showed a decrease. The PBS formulation showed a marked decrease in monomer content (Table 2H) at 25 and 40'C with a simultaneous increase in the HMMS content (Table 21) An exact opposite trend was observed for the sucrose (A and B) and trehalose (C and D) containing formulations where ar increased formation of monomer with simultaneous decrease in HMMS and dier content was observed at higher 25 temperatures. At 25C, both sucrose (A and B) and trehalose (C and D) formulations behaved similarly. At 40 C the trehalose formulations (C and D) showed significantly smaller changes in dimer, monomer and HMMS content compared to the sucrose (A and B) formulations at 40C* This demonstrated that at 40"C trehalose formulations are relatively more stable than the sucrose containing formulations.

Lyophzed formulations (TabLe 2G, 2H and 2i) All lyophitized formulations showed no/minimal changes at 5i 25 and 40QC at the end of 12 weeks except for lyophilized PBS (F) formulation. Percent Impurity and Shift in Retention Time by AEX 5 Liquid formulations (Table 2 J and 2K) At 5"0 (T=12". weeks) the percent impunity for both sucrose and trehalose forniulations was less than or equal to 1%. Retention time for the CDX-110 peak moved by approximately one minute for all formulations, The movement in retention time indicated formation of acidic species which has been attributed to a combination of 10 tinker hydrolysis and deamidation. At 25'C and 40'C (T=12 weeks) percent impurity increased significantly for all formulations, Change in retention time also followed a similar trend indicating increased formation of acidic species Retention time shifts for trehalose containing formulations was less compared to the sucrose formulations indicating less formation of acidic species in the trehalose formulations, 15 Lyophiized fomnulatons (Table 2V and K) For all lyophilized formulations, the percent impury change at all temperatures was not significant. A similar trend was observed for retention time (<I minutes) for all formulations Percent Impurity by RPHPLC 20 Liquid formuiaton (Table 2L) An increased formation of peoptide and linker related impurities were observed with increasing temperature for both sucrose and trehalose formulations. The trehalose formulation showed less formation of impurities than the sucrose formulations, Lyophiezad formulations (Tab/e 2L2 25 No significant change was observed for any of the formulations except for the lyoph ilized PBS (F) formulation. Mouse potency All liquid and lyophilized formulations (5*C samples) tested met the drug product specification of 75% seroconversion in mnice (Table 2M) Table 2D: Appearance Data Fo.ruaton Appearance at Appearance at Appearance at Appearance at =0 wk 5C T=2wk 250 T2wk 40CT=2wk NMA RSt NM() R NMO RM KP Sc drtess <'9 Cdess ('<BN Colatas ( 89' brown yEow mrgi E PFi I:VP EFVP FV< 1 sman white roud A ds ') Nyd 'Si MN?)$N RJi NMi R8! KP7 , u5o60 doss N (ASS le( Coons 9rs mMIA PC6 H shotwhit EFVP EV S(lig) NMO RS ~ NM?) R$ NMO RS NMC RSi NaP> Sucb' 8 CdPi<(9 Codosrks (<) CokAess 9 SM) &rwrm kM mg/ PS60 PH EF 1 white, 1 tan Eh00 EV(Yb) 7 -- (q fnarike Pate Fi <VP B (lye) NM) RSI NM?) R$ NMsRaMOR NaP?. Smc-SO Corleass (Y 9) Coviess (2 9) COkass (89) Cokaess (<89) m/ PS6), pH EFVP EFVP EFVP 1 shot white NMu RSI NMO R$ NMO RSJ NMO) R51 C q cdoress (CJ9 oloress (<oice"4s (n <9 Srown yaw Po4 Re 94 EFVP 1 shortwhit EFVP (<tY7) m S H oerkeparot e smnal wbum round T q and partlesi ev___ C (ly) NM?) RSI NMO R$ NMv RS5 NM? RS PO4ra CfrCess ( 89) Cdoriess0 (AP Corless BQ Coioss (<B9) rmg/'mi PS60 pH EFVP EFVP 2 mvwl e Wmto ndt 1FVP vNk0- RSI NMO R$ NMO RS NM0 RS Pu less (89) Celoress 89 v ight brwmn sligh brown KPO14 5e 90 M e* s w '?' fkekoe SPVP ('87) (87) mni mj PH pH 7A Hq mnd D) ((ye) NMUA RS1 T NMO RS NM RC NMOR KP"4 Tri-9 t ordess (<B9) Catorless B9) Coldess (mP9 Coloriss (<8B) mgm PS8 pH E-VP EFVP PP/P 2 ma9 whde tour d NM?) RS NMO RSI NM?) R31 NMO~ RS ess (,89 Cotoress 89) ColAyss (09) -5 Codiss (9 EFP EFP ,hortwhie fiber 2 wths lake i q) ,ke patita rfssls PBS, pH 7 4 lip .d5smal whtie PET PsH Tv \it0=5&MM 6 ma wi sa ke articles 1NR NM RS NM? RS NM) RS S"qghl rown Vers Slighc Vety Shghtly Verys Sgtty O( 8) Many'j -1 Bown 1 Brown (-8R 2 Brown Q) 3 shor Ibrown -'n ta sh'orttibed k shert vhste fibedlike tan short fiber? e fibers. Typicua of smd wie md 4 whito 2 omani White PaSpdr globar part -{a'blvmmr particls red 5 whste Xekaging 2 shnot Oc) (Toc) gkayir pacsis who' sl'9ke (los) EFvP: Essenhali iree of visib"e parscles NM0O Riflisl Not more opalescent than referee suspension'IAiPi - )T Table 2E: Change in Concentration Data Change in Change in Changei concentration concentration concentration o mqimL) at 5C in (mg/rnL) at 25C in (mg/mL) at 40C in 12 wks 12 wks 12 wks A '4 KPO Su 80 mg/m[ 0 14 0"04 0 35 FPS80, pH T74. li_________ A yo KPO.8 ullm 0, 08 0 03 0 13 NaPO Suc8 KPO4 Tre 4 m 0.01 0.03 0,07 230pH? 7A1f P-. e e4 mghi 0.03 0.02 0 09 KPO4, Tre-90 mg/mi, 40.01 0.00 0,02 PS80 pH 74, hig _________ __ ______ D itydo K(P0I 9re0g 02 0,04 0,02 p38 H 7.4. y~o 0,2 0,03 0.05 4' F yoH 4oT09 012 -0.45 Table 2F Change in A2801A340 Ratio -f-0-r -m -Ulation Chainge in Change in Change in A2&O/A340 at 5C in A280A$40 at 250 A280/A$40 at 40C 12 wks in 12 wks in 12 wks A (li) KP0.Snc-0 0W0' 0,45 0,-00 2380. PH 7.4. flq, _________ _________ A (yo) '(o Suc-8 mg/ink -0,02 0,01 0. 2 NaPO. Suc-80 mg/m PS80p H7421 0.45 0.21 B (yo) NaP0 4 Suc80 ing/mL PS80, pj74 ~0.02 0~6 0.06 C 0.o 01 2 79 5 65 Tfe-84 mgM P380, pH 7A lIq C tiyc) KPO4, Tre#4 mgmin 0.09 0,03 0.33 D1 0,3 3 0, KP4eT 90 mg/nt -001 2 95.45 rUo Tre.-90 mng/i 0,13 0.00 0.20 F 0,02 2,59 7.95 F034 -003 0,98 PBS. pH 7., yo Table 2G: Change in Percent Dimer Data Formulation Change in dimer % Change in dimer Change in dimer at 50 in 12 wks % at 25C in 12 wks % at 400 in12 wks A ~q K(PO Gu-$ s mq/gn-d -0,12 4. 08 -41,98 A flycf KPO80 mgmI 1.50 1.50 0 70 9330 p__490_________o_ Napo" S10.8 ,13 93 4573 mg/mf. PS80, pH 7A4 CR04, Im 'o4 -~..0 94.41 -14.61 .KP0-j- E- 4 m/nK, 1 50 .1-0.30 -4220 00 -18.40 mgm3P0s. pH 7 A , __________ _________ KPO4 Tm'4 mi. -0190 4 1 PS80p 43y -03 -2.35 265 F yNX' -01 0 -153360 -0 Table 2H Change in Percent Monomer Data Formulaion Change in % Change in % Change in % monomer at SC in monomer at 25C in monomer at 40C in 12 wks 12 wks 2ks P HO [ 0,24 4,64 2694 -PS li 7A l -9 Fom-ulation Change in % Change in % Change in % monomer at SC in monome at 25C i monomer at 40C n 12 wks 12 wks 12 wks A fOyo) KP0 4 Su80 m -020 -0.0 0.20 -9 ____P_____dt_ 3 Qiq) aPO. S-0 -092 5,52 35.32 mg mPS60pH 7A4 B Qyo) NaPO. Sue-80 -0,30 0,20 -0,30 KPO4. ro&4 mgqA 02 4.20 820 PS0p pH 7.Aq C Nyo K PO4 e 4 m 0806 o (bt~ X(0, Tre-B~ not, -0,2,0 80 98,pHT74. lyo KPO Tre 90 mint 070 4,10 11,30 250 pH 7A. Uq -P, 90 mgh 10 0.00 .0.30 F0.78 22832 tCL02 0 80 2, 0 -2. 70C PB&S pH ?.4J. 00 Table 2L Change in Percent HMMS Data Formulation Change in HMM$ Change in HMM$ Change in HMMS %at 5 in 12 wks % at 25C in 12 wks % at 400 in 12 W ks---- A pq) KPO Se00 mg 33 -2,63 -12.53 A Oyo) P Su mg-050 -040 NaP,27 -297 -12.97 mg/n PS80, pH 74 -1 (K3 C (g)q KP/Tr -04 2.36 -6.66 C, f yo) KPA Tre mg/ink -0.3 -0.40 0.60 PS803 pH A4 lyo P80 pH 74 4lq KPO4, Tre40Q gt 020 o0Ao 00 F (lyo) -0,30 '18,20 33 5 Table 2J, Change in Percent Impurities by AEX Data Formulation Change in AEX % Change in AEX % Change in AEX % impurities at 50 in impurties at 250 impurities at 40C 12 wks in 12 wks in 12 wks A t) KPO.. S80 mg/ 030 120 1,60 Siq)pH 7A. q NaP u-8020 0.0 1 0 mg/mPS80 pH 74 NPO8 PS 80 pH7 0F00(120 0M mg/m PS80 pH 7,4, 0. 1 A C (kig KP84T mgh 010 0.9 2.60 C Oyo) KPO4, Tre-84 mg/it, 020 0.10 0 P,* f80 pH 4 Iyo D (hiq} Kg 'e90 mg/ 1 00 270 350 0 (0N) KP04 T 90 mgik 1 01 0.50 040 p 0 90 90 3 30 F f Wyo) Example 3: Filtration Process Development 3. 1 Materia/s & Equipment The following process materials and equipment were used during this study: AKTA Cross flow tangential flow filtration unit from GE H ca 30K and 100K SHydrosart cassettes with 200 car membrane area (Sartorius # 308144590 2E -SG; 308144680 2ESG.); 30K Hydrosart cassettes with 0.1 m2 membrane (Sartorius # 308144590 2E-SG); Keyhole limpet hemocyanin (KLH) purchased as Vacmune (Biosyn Corporation lots 739690V1, R625258, 817873V1, and 817873V2); EGFRvIll peptide (Anbiopharm Inc. lot APi 080616); EGFRII peptide (Chinese Peptide 10 Corporation (CPC) lot # CH-0600453); Suifosuccinimidyi 4-[ mnaeimidomnethyl~cycohexane-carboxylate (Sulfo~SMCC) (Thermo, various lots, catalog # 22322); Dimethyl sulfoxide (DMSO), anhydrous 99:9% (Sigma Catalog # 276855), NaPh buffer = 100mM sodium phosphate pH 7.2; PBS 1 OmniPur 10x phosphate buffered saline diluted to 1x (VWR Catalog # EM6506): KPh buffer = 10mM 15 potassium phosphate pH 7,4; PETG (polyethylene terephthalate copolyester) bottles (various sizes from Nalgene). 3.2 Methods The overall process included, (1) an activation reaction between KLH and tinker; (2) an initial TFF step to remove excess liner and linker-reiated impurities; (3) a 20 conjugation reaction step between activated KLH and peptide; followed by (4) a second TFF step to remove excess peptide and peptide-related impurities and to change the buffer. An extinction coefficient of 1.38 for CDOX- 10 was used throughout this study, A more detailed description for each step follows below. 321 Activation Reaction Step 25 For most of the TFF runs, KLH (100160 mg) was aliquoted into a 15 mL or 50 mL plastic tube, diluted with NaPh buffer and water for injection (WFI), and mixed by inversion of the tube several times. The linker solution (pre.-dissoved either in water or DMSO) was added to the KLH mixture, gently rnixed by inverting the tube several times. and then hold stationary for the remainder of the 45 5 minutes. For runs 121161-165 30 and 121161>172 PETG bottles were used as reaction vessels and the solution was vigorously mixed with a stir bar during tinker addition and more gently mixed for the remainder of the reaction, Different activation reaction parameters such as iinkerKLH molar ratio, buffer concentration, linker stock solution concentration and reaction times were investigated as different process inputs into the TFF system, Complete details are provided in Figure 2A 3.2.2 First TFF Operation At the end of the activation reaction, the acfvated KLH solution was diluted with 5 NaPh buffer to 1,5-2,0 mg/mL protein concentration prior to being transferred through a transfer pump into the cross-flow system. Membranes were pre-sanitized before use. During the development and optimization of the TFF operation different parameters were irwestigated such as protein concentration during diafiltration, diafiltration buffer mnembrane molecular weight cutoff (MWCO) transmermbrane pressure {TMP), retentate 10 flow rate, and the number of turn over diafiltration volumes (TOVs), Complete details are provided in Figure 2E. 3.23 Coigation Step The diafltered activated KLH solution processed in the TFF system was recirculated at 10 mUmin and either held for a total of 255 minutes (from start of 15 activation reaction to the addition of peptide) or conjugated immediately with the peptide solution. Prior to the conjugation step, the protein solution in the reservoir was concentrated to 2 mg/mL in order to reach the target concentration for the conjugation reaction step, For all conjugation reactions, the target concentration for both KLH and peptide was 1,5 mg/mL. The peptide stock solution was pre-dissolved in NaPh buffer 20 and added in a single boLus directly into the TFF reservoir with a pipet. The KLH:peptide ratio was held constant at 1:1 (by mass ratio), Parameters investigated were peptide stock solution concentration and conjugation reaction time, During the reactions the TFF systern was set to recirculate at 0,5 L/minr with the permeate line closed and the retentate line wide open. Complete details are provided in Figure 2C, 25 3.2.4 Second TFF Operafian After completion of the conjugation reaction, a second diafiltration was carried out to buffer exchange the conjugated KLH and to remove excess peptide and pepide related impurities. For this second diafiltration step, the same membrane and the same operating conditions as the first diafiltration were used. Upon completion of the second 30 diafiltration, the retentate was removed from the TFF system and filtered using a 0.2 pim filter. A buffer rinse was performed across the membrane and collected separately. 3.2.5 Summary of Cross~ ow Expedment Figures 2A-2C summarize the key operating parameters for the ten development TFF runs completed to develop the optimized process, Runs 121161-090, 121161093, 121161-096, and 121161-100 were a set of initial runs to evaluate linkerLH ratio; tinker stock concentration and dissolution solvent, arnd peptide stock concentration Additionally, the MWCO of the membrane was evaluated (30 kDa vs. 100 kDaY The inain objective of runs 121161-120 121161-125, and 121161-130 was to 5 determine TFF operating parameters such as TMP, retentate flow rate, and flux, Run 121161-147 was a repeat of 121161-125, The reasons for repeating 121161-125 are discussed further below. Runs 121161-165 arid 121161-172 were demonstration runs of the process which was to be transferred to a piot plant, Run 121161-165 was done using a 160 mg charge of KLH and run 121161-172 was done using a 1 g charge of 10 KLH to assess the scalability of the optimized process. Example 4 Results and Discussion of Filtration Process Development Key analytical results for all the process development runs are summarized in 15 Figures 3A-30. Final drug substance was used for all assays, except for epitope density which used the retentate recovered from the TFF system before addition of excipients. It was unclear whether PS80. which is one of the excipients added, would interfere with the epitome density assay, Target ranges were based on results from previous cinical lots (i e, Lots 06-044-001 and 07-044-001, as shown below in Table 3A) 20 taking into account the normal variations in the assays. Nearly all the runs produced material which was on target for all assays or were at least close to targeted ranges. The only exception was % linker as determined by the AEX assay which was significantly higher in some lots compared to others. 25 Table 3A Lct ~SEC Peaks 1ASIC MRP Epitope ---- r- - - H Densiy QUQA42 lut 3 344 07-04- 1 10,2 8 5160 85 1 94a 93 W50 Runs 121161-0901, 3~06an 0 From runs 121161-090, -093, -096, and -100, several decisions could be made regarding the activation reaction. First, a decision was made after 121161900 to switch from a 100 kD to a 30 kD membrane. This decision was based on possible loss of activated KLH through the membrane. RP-HPLC analysis of the permeate seemed to 5 show some leakage of activated KLHt however, later wlk determined that the assay was prone to carry-over under certain conditions. The activated KLH seen in the permeate was most likely due to cary-over from previous runs on the column and not due to leakage through the membrane. Second. the decision was made to incorporate DISO into the process to provide 10 complete dissolution of the linker, thereby having more control over linkerKLH ratios. From these runs, it was determined that DMSO did not have a detrimental effect on product quatiy. The appropriate SEC profile was maintained and epitope density was close to the target range; however, the original 357A linker:KLH ratio (12116-090 & 121161-100) resulted in an epitope density which was just above the desired range and 15 100:1 resulted in too low of an epitope density (121161-093). Data from These runs and additional data from smali-scal activation experiments led to the decision to set the linker:KLH ratio at 200:1 A decision was also made to set the linker stock solution at 100 mg/mU The desire was to keep the concentration high to minimize the amount of DMS0 added to 20 the reaction, but at a concentration where linker dissolved quickly and easily. Run 121161-100 used a linker stock of 180 mg/mL but dissolution of the linker was not as quick as with 100 mg/mt The concentration of the peptide stock solution was set at 6 mg/mL This stock concentration provided the appropriate volume of peptide solution to be added to the 25 conjugation reaction which results in the desired peptide and activated KLH concentrations of 1,5-2,0 mg/mL without the need for further buffer addition. The final decision based upon these four runs was to set the NaPh buffer at 40 mM in the activation reaction. Runs 11i61420 *125, -130, anrw147 30 The purpose of the next set of runs was to determine the optimal values for TFF parameters such as TMP, retentate flow rate, and flux. By this time, formulation nomination had occurred and as a result, the second diafiltration buffer was changed for all runs going forward to KPh buffer, Unker:KLH ratio was set at 200;1 and linker was dissolved at 100 mg/mL in DMSO Run 121161-120 was set up to explore the lower 5 range of TMP and retentate flow rate. Assay results indicated that the material produced fulfilled all target specifications, except the % tinker as measured by AEX was significantly higher than seen previously. This increase in linker was orginally bought to be due to a decrease in retentate flow rate (50 vs. 100 mLlmin) between the initial set of 5 four runs and 121161-120 Run 121161425 was set up to address the hypothesis that a decrease in retentate flow rate was the cause of the decrease in linker dearance. The SEC profile showed that % diner was slightly out of the target range and epitope density, at 36 peptides/KLH, was close to the low end of the target range (30-64 peptides/KLH); 10 however, linker clearance improved significantly (0.3% vs IG% front 121161-120. For run 121161-130, a decision was made to try higher MWCO membrane (100 kD) to try to improve impurity clearance. At the same time, linkerKLH ratio was increased to 250:1 to increase the epitope density resulting in a peptide/KLH number more towards the middle of the targeted range. The resulting material fulfilled all 15 analytical targets, except for % linker which had increased to 34%, Since two parameters had been changed for this run (membrane MWCO and linker:KLH ratio) it was unclear what had caused the % linker to increase compared to run 121161-125. A decision was made to return to using a 30 kD MWCC nienmbrane since run 121161-130 had shown no advantage to using a 100 kD rnembrane 20 To address questions regarding the normal level of lot-to-lot variation in linkedr a decision was made to repeat run 121 16- 12 5 as closely as possible to determine if the low level of % linker could be achieved again, The results from this run showed that although the level of linker had decreased to 9%. it stilt wasn't as low as earlier runs (C 1%), To assess the true impact of these levels of linker impurities, calculations were 25 performed to determine a correlation between % by area and actual ppm. What was determined was that the levels of % tinker by area seen in the process development runs corresponded to < 3 ppm; thus, the actual amount of the linker impurities by mass is actually quite small and at an acceptable level. Runs 121161465 and -472 30 Run 121161-165 was a tab-scale demonstrations run using a 160 mg charge of KLH to test the acceptability of material made by the optiized parameters determined during process development; T these parameters are summarized in Figures 2A-2C and the analytical results shown in Figures 3A-3D, To build further confidence in the optimized process, a larger-scale lab run was carried out. This rn (121161-172) used a a I g charge of KLH. The TFF parameters are summrize in Figures 2A-20 and the analytical results in Figures 3A-3D. Parameters for scaling up the process to 1 g were based on keeping the flux constant (see TabIe 3B below) Also, n Table 3B are the scaleup parameters which can be used for running the process at a 10 g scale 5 Table 3B Parameter Units Bench Scate I gram scale- JJ Clinical up run Run Mass of KLH g 4.0 10 Memnbranr. n 0 02 12 Area VolumetArea Liter 65 35 6 56 Mass/Ae gr 100 3.3 Cross flowrate Un in 0 1 05 6 Gross flowrate LImintm5 5 5 per unit area Example 5- Reaction Parameter Process Development 10 52 MateriaLs and Methods The following process materials and equipment were used during this study: Keyhole m pet hemocyanin (,KLH) purchased as Vacmu ne@ (Biosyn Corporation lots 739690V1, R625258, 817873Vi and 817873V2) EGFRvlleptide (Ambiopharm Inc. lot APi 080616), EGFRvl peptide (Chinese Peptde Corporation (CPC) lot # CH 15 0600453); Sulfosuccinim idyl 4-[Nmalelmidomethylcydohexane-1 carboxylate (Sulfo SMO) (Thermo, various lots, catalog # 22322) Dimethy sulfoxide (DMSO), anhydrous 99,9% (Sigma catalog # 276835 or equivalent); NaPh buffer 100mM sodiuni phosphate pH 7,2; PBS = Gibco 1x PBS pH 7.2 (Giboo, catalog # 20012), or Dulbeccos 1x PBS (Gibco, catalog # 14190), or OmniPur 10x phosphate buffered saline diluted to 20 Ix (VWR catalog # EM6506); 2 mnL glass HPLC vals for reactions < 2mL (Atlent cat. no 5182-0715 or equivalent); 15 mL coming polypropylene tubes for reactions > 2 mL; Anmicon 50K and 100K Centrifugal Filters (Millipore cat no UFC805906 and UFC610096) or equivalent Zeba Desalt Spin Co umns, 0,5 ml (Thermo, catalog # 89883); Benchtop centrifuge.

$2 Methods The optinized manufacturing process includes: 1) an activation reaction which covalently lnks the amine groups of the KLH protein with the NHS-ester group of the Sulfo-SMGC linker to form activated KLH; 2) an initia TFF step to remove excess tinker 5 and linkerrelated impurities; 3) a conjugation reaction which covalently attaches the C> terminal cysteine of the synthetic EGFRvH peptide with the maleimide group of the Suifo-SMCC tinker which is part of the activated KLH molecule forming conjugated CDX-110, followed by, 4) a second TFF step to remove excess peptide and peptide related impurities and to exchange the buffer. This report describes the optimization of 10 reaction conditions using a scaled-down system called a one-pot reaction, An extinction coefficient of 1,30 for KLH and activated KLH and 1,38 for CDX-1 10 was used throughout this study. 52, Ono-Pot Reacfon KLH was aliquoted into a 2 mL glass HPL vialor a 15 mL polypropylene tube, 15 diluted with NaPh buffer, and mixed gently by version, The linked solution (pre dissolved either in water or DMSO) was added to the KLH mixture, vortexed briefly (15 30 sec) and then held for 45 * 5 minutes on an orbital mixer. After 45 minutes conjugation was started by quenching the reaction with peptide soktion at a ratio of 4'1 )eptideiinker and briefly vortexing (15-30 second) aton reaction was 20 allowed to react for -2 to 3 hours on an orbital mixer and then held overnight on the mixer or n a refrigerator prior to buffer exchange, Peptide and peptide-related impurities were removed by dialfltration with a. Millipore Anicon centrifugal filter prior to epitope density analysis Reaction parameters were adjusted as needed to investigate the desired variables such as inkerprotein ratio, activation time, protein concentration. 25 and percent DMSO. Each experiment is briefly described in the next section. Figures 4A-4C contain a summary of the reaction parameters that were used for each experiment, 522 Expeinent No, 122 123-065 KLH was activated with sulfo-SMCC at Iinker:KLH molar ratios varying between 30 100:1 and 357:1 for -45 minutes, Sulfo-SMCC inker was dissolved in DMSO at 8 mg/mL prior to addition. Peptide at a molar ratio of 242:1 (eptide:KLW) was immediately added to the reaction at the end of the acivation. The peptide:KLH ratio as was held at 22:1 and may not have been in an excess in the higher linker ratio experiments. An increased peptide'linker ratio was used in later experiments, 5.2.3 Experiment Nos 122123 309 122123-09 1221 2 12 KLH was activated with sulfo-SMCC at linker:protein molar ratios varying 5 between 100:1 and 440:1 for -45 minutes. Suffo-SMCC linker was dissolved in either water at 8 rng/mL or DMS0 at 30 mng/mL prior to addition. DMSO was not present in experiment 122123-097. DM80 varied between 3.5% and 15.6% for experiment 122123-109, and was held constant at 15,6% for experiment 122123-112. Peptide at a noiar ratio of 2.42:1 peptide:linker was inmediately added to the reaction at the end of 10 the activation for experiment 122.123-09e7 Although the results were acceptable, the peptidedinker molar ratio was increased to 4,1 for later experiments to ensure peptide was in excess. 5.2 4 Experiment No, 122123-115 Experiment 122123-115 examined percent DM80$ Sulfo-SMCCKL molar ratio. 15 and protein concentration using the full factorial Design of Experiments (DOE) shown in Figure 4C. Sulfo-SMCC linked was dissolved in DMS0 at 121.8 mg/mL prior to addition. Peptide at a molar ratio of 4:1 peptidelinker was immediately added to the reaction at the end of the activation, 5,2.5 E xperiment No. 12.2123 190 20 KLH was activated with sulfo-SMCC at inker:protein molar ratios varying between 25:1 and 350:1 for various activation times according to Figure 4A. SuWo SMCC linker was dissolved in DM8O at 100 mg/mL prior to addition. Peptide at a molar ratio of 4:1 peptide:linker was immediately added to the reaction at The end of the activation. 25 Example 6: Results of Reaction Parameter Process Development The focus of the reaction parameter process development was the optimization of reaction parameters using a scaled down system. The reaction volumes were less than 2 mL and clearance of peptide was achieved through the use of Amicon centrifugal filters. Clearance of peptide was needed in order to have an accurate analysis of 30 epitope density. The trends generated using the scaled-down reaction can be used to understand the effects and interactions of the process parameters but the clearance of peptide is not equivalent to a continuous TFF operation. The analytical attributes significant for reaction optimization are epitope density and size distribution profle. Purity of conjugates by AEX and/or RP-HPLC was used to ensure peptide was removed prior to analysis by amino add composition but is not included herein The targets for the optimized process are listed belowv in TAbie 4. 5 Table 4 Quality Targets for CDX-11O Drug Substance ParmeerMethod ____ Target ____ Epitope Density Aminc Acid 30-64 Pepides/KLH ______ ~Com ortian __________ Size Distribution SEC HPLC Peak I 2% Profile Peak 2 17% Peak 3 50-60% Peak 4 20-30% Peak 5 S% Purity AEX Peptide dimer 5% RPHPC ner or ker-related impurities 1% Figure 5 contains a typical SEC HPLC chromatogram for CDX-110 showing the 10 approximate retention times of the five peaks, Peaks I and 2 are believed to be high molecular weight species, peak 3 is believed to be dimer, peak 4 is believed to be monomer, and peak 5 is believed to be low molecular weight species. 6, 1 One-Pot ReacUanl The scaled down reaction model included testing multiple parameters at once 15 and used reaction volumes less than 2 mL. Although some experiments removed linker from the activation by slow diafiltration with Amicon filters, most exeriments used a onepot reaction model The one-pot system quenches the activation reaction and any tinker impurities with an increased molar ratio of peptide (4,1 peptiderlnker molar ratio) instead of clearing the linker from the reaction. Techniques to remove linker at this 20 small scale were either not effective at removing impurities or could change the SEC distribution during processing, - M70 The one pot reaction does not confound operation time with the variables tested and can show a true trend for the activation parameters However the SEC distribution results from the one-pot reactions are generally not always in the target range for the drug substance process because of the absence of a TFF operation time. The SEC 5 distribution and epitope density continue to change with time as shown for activated KLH after clearance of linker and for activation time (see 6,2) The effect of time on SEC distribution and epitope density was considered when selecting parameters for the optimized process 10 6.2 Molar Ratio of Sufo-SMCC:KLH and Activation Time Epitope Density The increase in epitope density associated with an increase in linker ratio for 15 experiment 122123-190 was fit to the second order polynomial shown in Figure 6, A second order curve has been used in preliminary kinetic oideling evaluations. This fitted curve can be used to predict epitope density at a given linker.protein ratio using the same activation conditions The results from experimerit 122123-065, also shown in Figure 6, are comparable to experiment 122123190, 20 A inker:protein ratio of 200.1, the ratio chosen for the optimized process, results in an epitope density of -45 peptides/KLH using a one-pot reaction scheme. The 200:1 ratio provides a mid-range epitope density and the entire target range of 30-64 peptides/KLH can be achieved by linker mnolar ratios of 200 ± 125 with a 46 minute activation time, The epitope density expected for the manufacturing process will be less 25 than this to account for the processing time to remove the linker by diafiltration. An activation time of -150 minutes resulted in a maximum epitope density of -60 peptides/KLH for a reaction using a molar ratio of 3501 Sufo-SMOC:KLH and a maximum epitope density of -60 peptides/KLH for a reaction using 2001 Sulfo SMCCKLH (Figure 7). After 150 minutes, the epit~ope density achieved at each linker 30 ratio begins to decline, The decline in epitope density with time could be related to hydrolysis of the adive naeimide or linker degradation. Similar epitope density curves result from both 45 minute and 4 hour activation times (see Figure 6). The similarity of the curves is related to the activation time results shown in Figure 7 where 45 minute and 250 minute (4 hour) have similar epitope - 71 densities. Higher epitope densities may be achieved at similar conditions if the same tinker ratio curve was generated at the maximum (150 min) SEC Distribution 5 Although more data points are needed to determine the curve shape the SEC results from experiment 122123'190 shown in Figures 8A and 8B were fit to a linear curve to show a general trend, Monomer species (peak 4) were shown to increase while high molecular weight (peak 2) and dimer species (peak 3) were shown to decrease with an increasing linkersprotein ratio, At the optimized process conditions, a 10 200.1 linkerprotein ratio and a 45 minute activation time, the SEC distribution does not meet the drug substance process targets because the reactions were performed using a one-pot reaction with no additional hold or processing time. Higher molecular weight species (peaks 1. 2 and 3) form over time during the activation reactions. This observation is consistent regardless of linker:protein ratio. 15 The SEC results for activation time (Figures 9A and 9B) were also fit to a linear curve to show a general trend although the actual relationships may not be linear. Results for peaks 1 and 5 are not presented but generally follow similar trends of more higher molecular weight species with decreased linkerprotein ratio and increased activation time; however, the percent of each species or the change in value is very 20 small, A straightforward relationship between epitope density and SEC heterogeneity has not been found. Each are affected by the same parameters but not necessarily in the same way, The parameters for the optimized process were chosen by considering how all the parameters interact, When the operation time for the TFF process is 25 combined with the changes in the SEC distribution over time, all of the SEC peaks and epitope density will be within the target range, 63 Disso/utton of Linker Sulfo-SMCC linker in a previous process was suspended in phosphate buffer 30 prior to starting the activation. The linker is not soluble in aqueous buffer at this concentration resulting in a linker slurry, The Sulfo-SMCC is fully soluble in DMSO at concentrations greater than 100 mgimL and will offer several advantages that will make the overall process more robust These advantages include: 1) Reduced linker hydrolysis - the linker will begin to degrade and hydrolyze as soon as it is in contact with aqueous solution but should remain stable in anhydrous DMS0, which will provide greater flexibility for manufacturing and 2) Better control of linkeripotein ratio the transfer and dissolution of a slurry is more difficult to control than an aqueous solution Small changes in the linker ratio could effect the epitope density and SEC distribution 5 significantly, Lnker Skuny vs Dissolution in DMSC Epitope density was similar for linker:protein ratios of 100:1 and 4401 when comparing linker added as a sluny (no DMSO) and tinker dissolved in DM30 (Figure 10 10): This observation is consistent vith the results shown in Figure 6 comparing experiments 122123-065 (no DMSO) and 122123190 (DMSO < 34%) which also had similar epitope densities for reactions with and without DMS0, DMSO added to the activation reaction was shown to reduce the amount of higher molecular weight species (Figure 1 IA and 11 B), Dimer (peak 3) and monomer 15 (peak 4) changed by 10-15% as DMSO increased from 0% to -15,6%, Peaks 1, 2, and 5 generally follow the same trends although the percent of each species or the change in value is very small. 64 DOE to Determine Effect of DMS0, Protein Corcentration, ano' Suli-SMCC Patio 20 The initial experiments to look at linker slurry showed almost no change in epitope density and a significant effect of DM3O on the SEC distribution. A follow-up experiment, 122123-115, was designed to further examine the effect of DMSO using a full factorial design of experiments (DOE) The experiment was designed to test DM30 (41 -156%), iinker:protein molar ratio (200:1 -357:) and protein concentration (5-15 25 ng/mLt Epitope Density An analysis of variance (ANOVA) of the epitope density results with a 95% confidence interval indicate that linker:protein ratio is a statistically significant factor. 30 Epitope density creases when more Sulfo-SMCC is present which is consistent with the results discussed in section 6.2. Although not a statistical interaction the final epitope density obtained for a given Sulfo-SMCC ratio is impacted by DMS and protein concentration At low DMO concentrations (4,7%), there is little effect o epitope density (Figure 12A), while at high DMSO concentrations (15.6%) epitope density is offset by approximately 10 peptides/KLH depending on the protein concentration (Figure 12B), If the confidence interval is reduced to 90%, protein concentration and the interaction between protein concentration and DM8O aso become significant 5 parameters effecting epitope density, At 4.7% DMSO, little change in epitope density is observed, At protein concentrations of 5 to T5 mg/mL, epitope density starts to increasee as the percent of DMSO in the reaction is increased. At protein concentrations of 12 to 15 mmL epitope density starts to decrease as the percent of DMSO in the reaction is increased. 10 SEC Distribution ANOVA of the dimer (peak 3) results with a 95% confidence interval indicate that iinker:protein ratio, percent DMSO protein concentrationand the interaction between percent DMSO and protein concentration are all statistically significant factors, Diner 15 (peak 3) decreases when more SulfoSMCC is present, which is consistent with the results discussed in section 62. Although not a statistical interaction, the percent diner obtained for a given SulfoSMCC ratio is impacted by DM O and protein concentration. At high DMSO concentrations (152%), there is little effect on epitope density (Figure 13$), while at low DMSO concentrations (43%) epitope density is offset by as much as 20 15% depending on the protein concentration (Figure 13A), The interaction effect between protein concentrating and DMSO on dimer (peak 3) can be summarized as folows. At 152% DMSO, title change in epitope density is observed. At protein concentrations of 5 to 11.5 mg/mL, the dimer (peak 3) starts to increase as the percent of DMSO in the reaction is lowered. 25 The protein concentration for the optimized process (10 mg/mL) did not change from the original process. At this concentration, the dimner (peak 3) changes slightly as the percent DMSO is increased from 4,7% to 15,6%. ANOVA of the monomer (peak 4) results with a 95% confidence interval indicate that Sulfo-SMCC ratio and percent DMSO are both statistically significant factors. Monomer increases when more Sulfo 30 SMOC and more DMSO are present, which is consistent with the results discussed in section 6.2.

65 Optimzed Process Figure 14 summarizes the reaction parameters used for both the original and optimized processes. The changes to the reaction conditions (highUghted in bold) are the following 1 Dissoluton of Sulfo-SMCC in anhydrous DMSO - reduced linker 5 hydrolysis and provided better control of Suifo-SMCC linked ratio; 2) Sulfo-SMCC molar ratio reduced to 200x - this change was to compensate for the dissolution of linker in DMSO; 3) Temperature - reduced to 1500 4) Peptide stock solution concentration increased to account for concentration of KLH after TFF operation. Since the peptde is soluble at >20 mg/ml and dimerization is slow, this should have little effect on the 10 reaction Example 7: Particulate Counts The U.S, Pharmacopeia <788> (USP <788> Particulat Matter in injections) sets forth certain criteria and protocolsregarding the measures nt of particulate counts per 15 vial. As indicated in USP <788>. for the determination of particulate matter, two procedures can be used: the Light Obscuration Particle Count Test: and the Microscopic Particle Count Test, The criteria set forth in USP<88> are as follows: particle size >10pm: not more than 6O00container; and particle size 25pm: not more than 600/container For CDX-110, a study was performed comparing two formulations 20 at time=0 and at the end of 26 weeks at 5"C to determine the effect of formulation components on particulate count using standard protocols as set forth in USP <788>. The first formulation (Formulation A) contained 1.0 mg/nL of the KLHEGFRvIII conjugate CDX-110 1.0 mg/mL, 10 mM phosphate buffer (Na.HPOdKH 2 P0.,), 137 mM NaCl and 2,7 mM KC The second formulation (Formulation B) contained 1'0 mg/mtL 25 of the KLH-EGFRviI conjugate CDX-110, 10 mM potassium phosphate buffer, 90njmL trehaose, and 02 mg/mL polysorbate 80, Table 5 and Table 6 list the particulate count for Formulation A at time 0 and after 26 weeks at 5'0, respectively. Table 7 and Table 8 list the particulate coont for Formulation B at time = 0 and after 26 weeks at 5*C, respectively The data 30 demonstrate no significant increase in particulate content at the end of 26 weeks for Formulation B. A significant increase in sub-visibile parties above 10 and 25 pm, however, was observed in 26 weeks for Formulation A when compared to Formulation B. Table S. Formulation A particulate count at time = 0 Particle Size (.in) Avg cumulative Avg cumulative particles/mL particles/container 1,5 926 555.6 2 366 219.6 56 3 6 10 6 3,6 15 1 0,6 20 0 0 25 0 0 Table 6: Formulation A particulate count at time 26 weeks, 5C Particle Size (jm) Avg cumulatitve Avg cumultie particles/mL particles/container 1.5 2401 1441 2 821 493 5 206 123 8 79 48 10 54 32 15 27 16 20 117 25 5 3 5 Table 7- Formulation A particulate count at time 0 Particle Size (jm) Avg cumulatitve Avg cumulative particles/mL particles/container 1.5 586 369 2 219 138 5 67 42 8 24 15 017 76 15 6 5 20 3 2 25 2 -' Table 8 Formulation B particulate count at time = 26 weeks, 5C Part cle Sze (tm) Avg cumulatitve Avg cumulative particles/mL particles/container 1,5 814 513 2 357 225 5) 64 40 8 20 13 10 12 8 is 5 3 25 2 1 SUMMARY OF SEQUENCE LISTING SEQ ID NO, DESCRIPTION---- SEQUENCE 1 LEGFRv Il peptt e LEKKGNYWTD LH 2 EGFRvm peptide with LEEKKGNYVTDHC C-terminal Cys residue for linking 3 E GFRvl i peptide wih CLEEKKGNYWTDH Nterminal Gys residue for linking 5

Claims (20)

1. A composition comprising a KLH-pepi de conjugate, a butter, a saccharide ard 5 a surfactant, wherein the peptide coniugated to KLH in the KLHpeptide conjugate comprises an EGFRvII amino acid sequence, 2, The composition according to ciaim 1, wherein the buffer comprises a phosphate buffer, 10

3. The composition according to claim 2, wherein the buffer comprises a potassium phosphate buffer, 4, The composition according to any one of claims 1 to 3, wherein the buffer is 15 present in a concentration ranging from 5 mM to 30 mM.

5. The composition according to any one of claims 1 to 3, wherein the buffer is present in a concentration such that after 12 weeks at 4WC, the concentration the KLH-peptide conjugate in said composition, as measured in mg/mL, has 20 changed by less than 15% when compared to the original concentration.

6. The composition according to claim 1, wherein the saccharide is a disaccharide. 7, The composition according to claim 6, wherein the disaccharide is trehalose, and 25 is present in a concentration ranging from 45 to 150 mg/mL or is present in an amount ranging from 80 to 110 mg per mg of KLH-peptide conjugate.

8. The composition according to claim 6, wherein the disaccharide is trehalose and is present in a concentration such that after 12 weeks at 40'C, the concentration 30 of the KLH-peptide conjugate in said composition, as measured in mg/mL has changed by less than 15% when compared to the original concentration. 7s

9. The composition according to claim 1, wherein the surfactant is polysorbate, and is present in a concentration ranging from 0.01 to 0.3 mg/mL or is present in an amount ranging from 0.01 to 0.3 rug per mg of KLH-peptide conjugate, 5 10. The composition according to claim 1.vherein the surfactant is polysorbate and is present in a concentration such that after 12 weeks at 40'C, the concentration of the KLH-peptide conjugate in said composition, as measured in mg/til, has changed by less than 15% when cornpared to the original concentration. 10 11 The composition according to any of the preceding claims wherein the peptide conjugated to KLH in the KL H-peptide conjugate consists of SEQ lD NO:1. 12, The composition according to any of the preceding claims, wherein the peptide conjugated to KLH in the KLHi-peptide conjugate consists of SEQ ID N0:2. 15

13. The composition according to any of the preceding claims, wherein the peptide is conjugated to KLH with a sufo-SMCC linker, 14, The composition according to any of the preceding claims, wherein the epitope 20 density ranges froi 20 to 80,

15. The composition according to any of the preceding claims herein the amount of KLH-peptide conjugate present in dimer form ranges from 45% to 65% by mass of the total mass of the composition, as determined by size exclusion 25 chromatography.

16. The composition according to any of the preceding claims wherein the amount of KLHpeptide conjugate present in monomer form ranges from 15% to 40% by mass of the total mass of the composition, as determined by size exclusion 30 chromatography.

17. The composition according to any of the preceding claims wherein the amount of KLH-peptide conjugate present in monomer form ranges from 18% to 35% by mass of the total mass of the composition, and the amount of KLH-peptide conjugate present in dimer form ranges from 50% to 65% by mass of the total mass of the composition, as determined by size exclusion chromatography,

18. The composition according to any of the preceding claims, wherein said 5 conmpositon is an aqueous pharmaceutical composition. and the pH of said composition ranges from 6 to 8,

19. The composition according to any of the preceding claims, wherein said composition is an aqueous pharmaceutical composition, and the pH of said 10 composition is an amount such that after 12 weeks at 40'C the concentration of the KLH-peptide conjugate in said composition, as measured in mg/mL has changed by less than 15% when compared to the original concentration,

20. A liquid composition comprising a KLWEGFRvill peptide conjugate, potassium 15 phosphate buffer, trehalose, and polysorbate 80, wherein: the peptide conjugated to KLH comprises SEQ ID NO:1: the KLHKEGFRvil peptide corjgate has an epitome density ranging from 30 to 65; the buffer is present in a concentration ranging from 9 mM to 11 mM H the pH otecomposition ranges fIon 7. to i5 the trehalose is present in a concentration ranging from 85 mg/mL to 95 mg/mt 20 the polysorbate 80 is present in a concentration ranging from 0.1 m/nmL to 0.3 mg/mL; and further wherein the amount of KLH-EGFRvIll peptide conjugate present in monomer form ranges froi 18% to 35% by mass of the total mass of the composition, and the amount of KLH-EGVRvI peptide conjugate present in dimer form ranges from 50% to 65% by mnss of the total mass of the 25 composition, as determined by size exclusion chromatography, 21, The composition of claim 17, wherein the liquid composition is prepared by reconstituting a lyophilized composition with water. 30 22. The composition of claim 17 or 18, wherein the buffer is present in a concentration of 10 mM, the pH of the sompostion is 7.4, the trehalose is present in a concentration of 90 mgl/L, and the polysorbate 80 is present in a concentration af 0,2 mg/mL 23, A method for preparing a KLH-EGFRvIiI conjugate comprising:a) combining KLH with a linker and allowing the KLH and linker to interact for a time ranging from 30 to 60 minutes; and b) adding a peptide comprising SEQ ID NO: I to the activated KLH product resulting from step a) to provide the KLH~EGFRvII Conjugate. 5

24. The method of claim 20. wherein the linked is combined with KLH in a linker:KLH molar ratio that ranges from 751 to 325:1. 25, A method for preparing a KLHEGFRvilI conjugate comprising: a) combining KLH 10 with a linker and allowing the KLH and linker to interact; and b) adding a peptide comprising SEQ ID NO: I to the activated KLH product resulting from step a) to provide the KLH-EGFRvyli conjugate, wherein the linker is a sufo-SMCC linker which is added in a non-aqueous solvent 15 26. A method as claimed in claim 25 wherein the non-aqueous solvent comprises DMSO 27, A composition comprising a KLH-EGFRvlll peptide conjugate wherein the arnount of KLI~EGFRvliI peptide conjugate present in mornome r fon ranges 20 from 18% to 35% by mass of the totat mass of the composition, and the amount of KLH-EGFRvili peptide conjugate present in dimer form ranges from 50% to 65% by mass of the total mass of the composition, as determined by size exclusion chromatography. 25 2& A composition comprising a KLH-EGFRvlll peptide conjugate wherein said composition is an aqueous pharmaceutcal composition which has been sta bilised such that after 12 weeks at 40C, the concentration of the KLH EGFRvIII peptide conjugate in said composition, as measured in mg/mL, has changes by less than 15% when compared to the original concentration. 30

29. A composition comorising a KLH-EGFRevI peptide conjugate wherein the m an epitopC density of said conjugate is between about 20 and 80.

30. A composition comprising a KLH-EGFRvill peptide conjugate wherein said composition has the following properties i) epitope density as measured by armino acid composition of 30-70 5 peptidesKLHt; ii) size distribution profile as measured by SEC HPLC as follows: Peak I <2% Peak 2 9-17% 10 Peak 3 50-60% Peak 4 20-30% Peak 5 1-5% iii) purity as measured by AEX RPHPLC as folloivws Peptide dimer < 5% 15 Bunker or linker-related impurities s 1% Total peptide-iinker impurities 10%, 31, The composition according to any one of cils27 to 30. which comprises a 20 buffer.

32. The composition according to daim 31 wherein the buffer comprises a phosphate buffer, 25 33 The composition according to cairn 32, wherein the buffer comprises a potassium phosphate buffer.

34. The composition according to any one of claims 27 to 33 which comprises a croprotectant or lyoprotectant 30 35, The composition according to any one of claims 27 to 33 which comprises a sacchaide. 36 The composition according to claim 35 wherein the saccharide is a disaccharide 35

37. The composition according to claim 36 wherein the diaccharke is trehalose, 82 38 The composition according to claim any one of claims:27 to 37 which comprises a surfactant 5 39. The composition according to daim 38 Wherein the surfactant is a polysorbate, poioxamer or triton surf actant,

40. The composition according to daim 39 wherein the surfactart is polysorbate 80 10 41 The composition according to any one of claims 27 to 40, wherein after 26 weeks at 5*C, the number of particulates greater than or equal to 25 pm, as measured by USP <788> increases by less than 1000%. 15