WO2001014884A1

WO2001014884A1 - Melanoma associated antigen (hmw-maa) defined by a monoclonal antibody

Info

Publication number: WO2001014884A1
Application number: PCT/SE2000/001597
Authority: WO
Inventors: Thomas N. Brodin; Lennart G. Ohlsson; M. Jesper Tordsson
Original assignee: Active Biotech Ab
Priority date: 1999-08-24
Filing date: 2000-08-22
Publication date: 2001-03-01
Also published as: AU762240B2; NZ517132A; CA2381099A1; EP1206702A1; SE9902989D0; AU6883700A

Abstract

An epitope of high molecular weight melanoma associated antigen (HMW-MAA) displayed on the surface of human cells but not cells from Macaca fascicularis is described, said epitope a) having the ability of being specifically blocked by and specifically block a binding structure which recognizes HMW-MAA, b) being displayed on the surface of human cells but not cells from Macaca fascicularis, and c) having a normal tissue distribution pattern which is more restricted than that of other known epitopes of HMW-MAA. There is also described a binding structure and antibodies recognizing target structures of said antigen, pharmaceutical compositions and vaccines as well as methods of using said epitopes and antibodies in diagnostic and therapeutic methods related to human malignant diseases.

Description

) to t o σι o LΠ O

CD 0 πr o rr tr rt rt 3 H- CO n <! n 0⁾ O 0 TJ Ω CD rt Ω 3 ω 3 CD TJ rt 2 πr

CD H- 0 C SD 0 £ O SD Hi rt SD H- *<_ C Hi SD c (D πr (D O > CD TJ O SD 5 H-

P 3 3 H p. 3 <. ii 3 rt rt rt ^yQ CD rt H H H- I-¹ ii n rt H- CD H [ IQ rr H- 3 0 SD CD H- O (D H- O SD ii O CD yQ H- CD co D « πr rt H- ID πr co n- fl) ID H fl) fl) ϋ c LQ h-¹ rt cr O rt ii CD O fl) ^ Ω 0 O rt CD fl) fϋ rt CO u. H Hi <J H- fl> O CD HI O rt H fd TJ H- rt 3 t-3

S α SD 3 s; fl) H 3 3 SD fl) H! SD X CD rt rt s: CO H- ii πr πr o to CD O SD 0 πr

3 fl) fl> p. i fl) fl) 3 HI 3 HI H- c rt CD H- rt CD SD CD Cj CO P CD h-¹ CD

=> 3 0 0 H- SD CO HI C- O fl) ro o H H- πr rt SD CD SD p ^ ri CD rt cr CD

CD o 3 c iQ p. P- TJ rt Hi H- HI P rt IQ 3 CD (D πr CD SD H- σ CD SD H- Ω TJ

H P⁾ to πr fl) co 0 O fl) rt SD fl) SD H CD 3 CD CD CO rt HI HI SD C Hi M rt c

3 rr fl) Hi O CO rt n HI H C 3 < CO CO CD c ≤ Ω o SD P HI Ω (-" CD

SD o ^ H- CO ii πr • rt rt H- SD CD ct-

O C rt rt O ^ HI 0 SD c Ω 0 H- _ lf) ri⁾

3 <_ fl) fl) ii CD H- CD O CO H O H- Ω CD SD C Hl ii CD Q.

0 t-¹ P⁾ fl) fl) 0⁾ •> J i SD cr 0 rt Ω ID Cfl CD CD H H⁾ < H ID HI

Ω rr Hi H to SD πr 0 O fl) fl) to 0 Hi K rt SD CD s; rt J c H- ϋ SD ^•<; ø^j 0 rt TJ 0 H- O ID. H- & H 3 rt CO h-¹ Ω t rt H- Ω CO CD CD D

3 co h-¹ Hi H- !-⁾ fl) CD CD w Hl H- 3 H- 3 SD

^ πr CD O cr Ω rt H- H-

CD CQ H- to O 0 !-J ϋ c rt CD CD ^ 0 Ω 3 H- h- ' CD c H 0 H- O i IQ ΠJ σq

HI C 3 rt 3 CO rt 1 ^yQ 3 CO πr H SD C H⁾ H- O g πr ΠJ C πr <: rr fl) fl) h SD rt 3 T H- 3 fl) H- fl) h-¹ rt s: iQ TJ c ^ c H- CD Ω rt CD 3

0) co 0 fl) fl) 0⁾ O fl) CD SD O O O πr H3 0 rr ii M 3 CD CO to rt Hi

P HI ⁾ 3 rr ii CO SD πr SD 3 H- SD H- i μ- 3 S (D CD SD C 3 rt

H- SD IQ CO fl) HT rt J H- CO HI SD P. O 0 P h-¹ HI HI CD 3 SD H HI SD H- H CD H- 3

H- co !-J h-¹ CD fl) _-^■ O H- TJ (D H- O H- CD < ω H- CD rt rr (D CD H H- CO P CD O

13 rr ^ 0 n SD O TJ rt 0 3 Hl TJ SD SD H- CQ rt ¹- o 3 πi SD HI

3 i o rt Hi CO C ϋ fl) HI SD CO fl) Hi Ω CO rt 3 0 3 rt ⁽D ≥! SD s: (D (D HI

C H- rt fl) H- o 0 O rt Ω Ω H- SD CD πr CD CD πr HI CD >

HI HI o H- >

!-⁾ cr H- . 0⁾ ϋ 3 s: SD SD O TJ H- fl) rt 3 CD Hi CD SD H- H- r 3 CO

0 C- co T rr J HT ii o 3 SD rt H- h- ' H- SD rt rt HI IQ SD H- SD h- ^■ rr to i fl) 3 H- h- ^■ πr CD rt c H- (D ^•<; 3 O HI H- rt HI ID IQ SD ϋ α>

0 H- C fl) p. 0 0⁾ O ^ CD O rt H- 3 Ω CO CO SD c O 3 O SD SD HI SD CD HI SD CD Q O fl) to CO HT CD πr πr fl> 0 SD rt SD rt H 3 CD CD 3 HI 0 CD y rt CO

H- ns to {" fl) to rt SD fl> HI H SD Ω CD <! SD rt CD rt CD H- CD SD o • 0) fl) Hi n 0 C HT H- HI rt ID. (D rt H- TJ rt 3 cr O rt

SD TJ rt £ Ω CO fl) Hl H- TJ CD SD ^ CD CO H- < . Ω O SD SD H- CD TJ 0 Ω fl)

H fl) H- H- fl) n CQ i • CD < (D SD ii rt o Hi H- Ω rr πr H- πr n H- ω fl) SD 1-3 3 H- CO TJ SD CD H- HI H- O CD πr (D H- SD rr (D H- ΠJ ro CO rt 3 1 C CO 3 CD O ^ H co CD ^ 3 CD SD 0 CD rt rt

SD 3 P • fl) O O a SD H- o CD H- CD CD H- cr H- HI CD SD SD HI 3 to CD O

H <D fl) 0⁾ - C Hi fl) fl) H- i H⁾ H- CD 3 Si O SD CQ CD SD rr SD CD P CD SD Q !-⁾ HI H 0 HJ ^■ ; U3 SD SD <. CO I CD H πr CD Ω Hi SD fl) rt to πr ffi πr 0⁾ o 3 h- ' rt rt H SD H- CD TJ CD HI CD SD CD rt 0 CD CD SD HI a rr H- H- s fl) Hi fl) 3 rt fl) CD C ^ Hi • O O CO . CD . πr c Ω P o

LQ 0⁾ ω ≤ rt 0 cr rt fl) (.: 3 rt O CD C CD rt O rt fϋ

3 fl) P πr 1 πr H- Hi 0⁾ πr 3 O O rt rt 3 H H- H- 3. H- ii c H- ID H- TJ o

0 3 P. 2 H- ua <D ω 0 SD 0 SD SD H C rt ς. CD Ω co O πs (D to Ω HI ID H- D ua A) cu H⁾ h CD HI 3 πr 3 P- fl) CD Ω TJ H- SD CD rt P

CD i πr ϋ [p πr ns α CD O CD rt Ω O CD O (D 0 CD H rt H CD πs ts] HJ O

O fl) SD fl) •• CO 0⁾ 3 CD CD H- CD H H i HI H 3 to H- CD c ID H- T P3

C T to £) 3 Hi ØJ 3 SD s^ C£! CO SD CD O rt H- πι Ω SD rt Ω

. Hl _^-_^ CD 3

H £ H- 0 rt H⁾ fl) ID. P. CD TJ 3 πι ID i O H- to O ID πα ct fl) fl) 0⁾ fl) CD 0 H- rt SD *< SD ^ Ω H- CD Ω SD H-

U3 CD HI CO

1 fl) 3 O H 1 Hi fl) 3 CD πr CD rt H- 1 Hi n O

1 1 rt CO 0 H- H- H- H H- 1

0 ^ rt HI Hl Ω co o 1 =<;

1 πr 3

10 and beyond can be examined efficiently by the antigen driven selection principle. Phage selection methods based on complex antigens such as cell suspensions have been described. Recently, a method to select antibody phage using tumor tissue sections was described. Taken together these methods enable the identification of novel antibody specificities against both in vi tro and in vivo expressed antigens. BRIEF SUMMARY OF THE INVENTION The present invention is based on the identification of new epitopes on the high molecular weight melanoma associated antigen.

Thus, the invention provides in a first aspect an epitope of high molecular weight melanoma associated antigen (HMW-MAA) , said epitope a) having the ability of being specifically blocked by and to specifically block a binding structure which recognizes HMW-MAA, b) being displayed on the surface of human cells but not in cells or tissue from Macaca fascicularis, and c) having a normal tissue distribution pattern which is more restricted than that of other known epitopes of HMW-MAA.

In one embodiment of said epitope the binding struc- ture is labeled and the binding thereof is inhibitable by an unlabeled form of said binding structure and not by other binding structures, and not inhibiting the binding of other binding structures having other specificities. In another embodiment of said epitope, said binding structure comprises one or more of the CDR (complementarity determining region) sequences comprising the amino acids number 23-33, 49-55, 88-98 and the amino acids number 158-162, 177-193, 226-240 of the amino acid sequence shown in SEQ ID NO: 2, the amino acids number 22-32, 48-54, 87-97 and the amino acids number 157-161, 176-192, 225-239 of the amino acid sequence shown in SEQ ID NO: 4, or other binding structures having essemtially the same epitope specificity.

By "essentially the same specificity" is meant that the binding of a binding structure structure (e.g. an antibody) to the epitope can only be blocked (binding inhibited) by the same binding structure or other binding structures having essentially the same specificity, and not by other binding structures having other specificites . Furthermore, the binding structure having essentially the same specificity should not block the binding of other binding structures having other binding specificites .

The binding of the binding structure (e.g. antibody) and other binding structures of essentially the same specificity could also be blocked (binding inhibited) by a soluble form of the epitope, but not by other epitopes.

In a further embodiment of said epitope, said binding structure is an antibody.

In a still further embodiment of said epitope, said antibody comprises the variable region of a light chain comprising essentially the amino acids number 1-109 of the amino acid sequence shown in SEQ ID NO: 2, and the variable region of a heavy chain comprising essentially the amino acids number 128-251 of the amino acid sequence shown in SEQ ID NO : 2.

In yet another embodiment of said epitope, said antibody comprises the variable region of a light chain comprising essentially the amino acids number 1-108 of the amino acid sequence shown in SEQ ID NO: 4, and the variable region of a heavy chain comprising essentially the amino acids number 127-250 of the amino acid sequence shown in SEQ ID NO : 4.

In another aspect of the invention, a further characteristic feature of said epitope is that it is display- ed less in human uterus and kidneys than other epitopes of the HMW-MAA. In a further aspect of the invention, there is provided an anti-idiotype of said epitope, which anti-idio- type is specifically blocked by and specifically blocks a binding structure having essemtially the same binding specificity for said epitope.

In another aspect of the invention, there is provided a vaccine composition comprising as an active principle an epitope as defined above, or an anti- idiotype of said epitope as defined above. A further aspect of the invention relates to a binding structure which recognizes an epitope of the high molecular weight melanoma associated antigen (HMW-MAA) and is of an organic-chemical nature.

Examples of such binding structures are antibodies and so called peptidomimetics . Said peptidomimetics are small organic-chemical drug-like molecules which can be derived from a polypetide, such as an antibody, by determination of the three-dimensional structure of e.g. the CDR loops in an antibody combining site. By such an elucidation of the bioactive conformation of a peptide loop in an immunoglobulin VL domain, so-called peptidomimetics based on organic-chemical molecules can be identified/constructed (6, 7, 8) .

A still further aspect of the invention is related to an antibody having a binding structure which recognizes a target structure of high molecular weight melanoma associated antigen (HMW-MAA) , and which comprises a) the CDR sequences in the light chain comprising essentially the amino acids number 23-33 (CDR1) , 49-55

(CDR2) and 88-98 (CDR3) of the amino acid sequence shown in SEQ ID No. 2, and the CDR sequences in the heavy chain comprising essentially the amino acids number 158-162 (CDR1) , 177-193 (CDR2) and 226-240 (CDR3) of the amino acid sequence shown in SEQ ID NO: 2, or b) the CDR sequences in the light chain comprising essentially the amino acids number 22-32 (CDR1) , 48-54 (CDR2) , 87-97 (CDR3) of the amino acid sequence shown in SEQ ID NO : 4 , and the CDR sequences in the heavy chain comprising essentially the amino acids number 157-161 (CDR1) , 176-192 (CDR2), 225-239 (CDR3) of the amino acid sequence shown in SEQ ID NO : 4 , or other binding structures having essemtially the same epitope specificity.

In an embodiment thereof, said antibody is phage selected. In another embodiment of said antibody the CDR sequences are of Macaca fascicularis origin.

In another embodiment of said antibody, the CDR sequences have an identity of at least 89% to corresponding CDR sequences of human origin. In another embodiment said antibody has low immuno- genicity or non-immunogenicity in humans.

A still further embodiment of said antibody is an antibody which has been derivatised by genetically linking to other polypeptides, or by chemical conjugation to organic or non-organic chemical molecules, or by oligo- or multimerisation. Also contemplated is an antibody, which is genetically linked or chemically conjugated to cytotoxic polypeptides or to cytotoxic organic or non-organic chemical molecules, or to biolo- gically active molecules, or to immune activating molecules .

Said antibody may also be changed to increase the affinity thereof, or to increase the production yield thereof, or to influence the pharmacokinetic properties thereof, or to give new pharmacokinetic properties thereto.

In a further embodiment, said antibody is labeled and the binding thereof is inhibitable by an unlabeled form of said antibody and not by other binding struc- tures, and not inhibiting the binding of other binding structures having other specificities. In one of its aspects, the present invention relates to a pharmaceutical composition comprising as an active principle an antibody as defined above.

In a further aspect, the invention relates to a method of in vitro histopathological diagnosis and prognosis of human malignant desease, in which a sample is contacted with an antibody as defined above and an indicator.

According to some embodiments, said method comprises tumor typing, tumor screening, tumor diagnosis and prognosis, or monitoring premalignant conditions.

In a still further aspect, the present invention relates to a method for in vitro diagnosis and prognosis of human malignant disease, whereby concentrations in bodily fluids of an antigen, comprising an epitope as defined above or an anti-idiotype of said epitope as defined above, is assayed.

In yet another aspect, the present invention relates to a method for in vitro diagnosis and prognosis of human malignant disease, whereby concentrations in bodily fluids of an antibody as defined above is assayed.

In still another aspect, the present invention relates to a method for in vitro diagnosis and prognosis of human malignant disease, whereby concentrations in bodily fluids of a complex of a) an antigen comprising an epitope as defined above or an anti-idiotype of said epitope as defined above, and b) an antibody as defined above is assayed.

In a further aspect, the present invention relates to a method for in vivo diagnosis and prognosis of human malignant disease, whereby the localisation of an antibody as defined above to tumor deposits in a human subject is determined.

In a further embodiment, said antibody is adminis- tered to the subject before the determination. In another embodiment, said antibody is accumulated in tumor deposits . In a further embodiment, said method is quantitative .

A still further aspect of the present invention is related to a method for therapy of human malignant dis- ease, whereby an antibody as defined above is administered to a human subject. In an embodiment of said method, said antibody has been changed by being genetically linked to molecules giving the combined molecule changed pharmacokinetic properties. In a further embodiment, said antibody has been changed by being derivatised.

The invention also encompasses DNA sequences coding for said amino acid sequences, which DNA sequences are shown in SEQ ID NOs : 1 and 3. DETAILED DESCRIPTION OF THE INVENTION

The high molecular weight melanoma associated antigen, HMW-MAA, has been demonstrated to be useful for diagnosis and treatment of malignant melanoma (2,3). Murine monoclonal antibodies (mAbs) generated towards different epitopes of this cell-surface molecule localize efficiently to metastatic lesions in patients with disseminated disease. The use of a phage selected antibody directed to this molecule for targeting of bacterial superantigens (SAgs) and cytotoxic T cells to melanoma cells has now been explored.

Macaca fascicularis monkeys were immunized with a crude suspension of metastatic melanoma. A strong sero- logical response towards HMW-MAA was demonstrated, indicating its role as an immunodominant molecule in the primate. By phage selection using melanoma cells and tissue sections of a recombinant antibody phage library generated from lymph node mRNA, several clones producing monoclonal scFv antibody fragments reacting with HMW-MAA were identified. One of these scFv fragments, denoted K305, was transferred to a Fab format and evaluated as the tumor targeting moiety for superantigen-based immu- notherapy. It binds with high affinity to a unique human- specific epitope on the HMW-MAA, and demonstrates more restricted crossreactivity with normal smooth muscle cells than previously described murine mAbs . The K305 Fab was fused to the superantigen Staphylococcal Enterotoxin A (D227A) , mutated to reduce its MHC Class II binding affinity and demonstrated redirection of T cell cytotoxi- city to melanoma cells in vi tro . In SCID mice carrying human melanoma tumors, engraftment of human lymphoid cells followed by the K305 Fab-SEA (D227A) fusion protein, induced HMW-MAA-specific tumor growth reduction.

The phage selected K305 antibody demonstrates properties including high affinity and selectivity supporting its use as a therapeutic antibody in conjunction with T cell activating superantigens . An antibody phage library derived from a tumor- immunized Macaca fascicularis monkey was used for phage selection using melanoma cells and tissue sections. The approach was based on previous reports that primate antibody repertoires have been used successfully for the pro- duction of selective immune sera to tumor-associated antigens. Several monoclonal Macaca fascicularis antibodies were selected which were specific for the HMW-MAA. One of these antibodies, here denoted K305, which together with K461 recognize a unique and selectively expressed epitope on the human HMW-MAA molecule, was fused as a Macaca fascicularis Fab fragment to SEA(D227A) . T cell lysis of melanoma cells and in vivo tumor reduction mediated by this antibody are demonstrated suggesting its applicability for immunotherapy of malignant melanoma.

The high molecular weight melanoma associated antigen, HMW-MAA, has been demonstrated to be useful for diagnosis and treatment of malignant melanoma. Murine monoclonal antibodies (mAbs) generated towards different epitopes of this cell-surface molecule localize efficiently to metastatic lesions in patients with disseminated disease. Now, the use of a phage selected antibody directed to this molecule for targeting of bacterial superantigens (SAgs) and cytotoxic T cells to melanoma cells has been explored. Macaca fascicularis monkeys were immunized with a crude suspension of metastatic melanoma. A strong sero- logical response towards HMW-MAA was demonstrated, indicating its role as an immunodominant molecule in the primate . By phage selection using melanoma cells and tissue sections of a recombinant antibody phage library generated from lymph node mRNA, several clones producing monoclonal scFv antibody fragments reacting with HMW-MAA were identified. One of these scFv fragments, here called K305, was transferred to a Fab format and evaluated as the tumor targeting moiety for superantigen-based immu- notherapy. It binds with high affinity to a unique human- specific epitope on the HMW-MAA, and demonstrates more restricted crossreactivity with normal smooth muscle cells than previously described murine mAbs. The K305 Fab was fused to the superantigen Staphylococcal Enterotoxin A (D227A) , mutated to reduce its MHC Class II binding affinity and demonstrated redirection of T cell cyto- toxicity to melanoma cells in vi tro . In SCID mice carry- ing human melanoma tumors, engraftment of human lymphoid cells followed by the K305 Fab-SEA (D227A) fusion protein, induced HMW-MAA-specific tumor growth reduction.

The phage selected K305 antibody demonstrates properties including high affinity and selectivity suppor- ting its use as a therapeutic antibody in conjunction with T cell activating superantigens.

The invention is further illustrated in the following nonli iting experimental part of the description. EXPERIMENTS Materials and Methods

Animals and immunization procedures

Severe Combined Immunodeficient (SCID) female mice (C.B-17) were obtained from Bommice, Ry, Denmark and kept under pathogen-free conditions. The animals were acclimatized to laboratory conditions (20 ± 1°C, 50 + 5% relative humidity) for about one week before commencement of the experiments. The animals were subjected to visual inspection on arrival and housed in Macrolone cages (III) with 10 animals in each cage. After randomizing, the animals were used for the experiments. The mice were fed sterile pelleted rodent diet from Special Diets Services, Essex, UK. Sterile water was always available ad libitum. The mice were 8-12 weeks of age when used in this study and the body weight ranged between 17.0-26.0 g with a mean value (±SEM) of 20.6±0.15 g.

Macaca fascicularis monkeys (two individuals) were kept and immunized at the Swedish Institute for Infec- tious Decease Control (SIIDC) , Stockholm. The monkeys were immunized s.c. with 2 ml of a crude mechanical suspension of 43 different metastatic melanoma tissue biopsies in PBS with 10 % normal Macaca fascicularis serum mixed with alun adjuvant. Booster doses were given day 21, 35, and 49. The immune serum was collected day

56. The large number of melanoma samples was used to select for an antibody response to commonly expressed melanoma associated antigens. Tissues and cells Human tumors and normal tissue samples and peripheral blood mononuclear cells (PBM) from blood-donors were obtained from Lund University Hospital and Malmδ General Hospital, Sweden. The PBM were isolated by density centrifugation over a Ficoll-Paque cushion (Pharmacia, Uppsala, Sweden) . The human melanoma cell lines FM3 and FM55 were a kind gift from Dr. Jesper Zeuthen while FMEX and G361 were from the American Type Tissue Culture

cells with 50 μl 33 μg/ml Genenase for 15 min. The cells were removed after centrifugation and 250 μl 1% BSA-PBS added to the supernatant. The phage titers were determined by infecting bacteria and counting colony forming units using the bacterial strain E . Col i DH5aF'. Phage rescue and culture of the phage library were according to standard techniques.

Production of SEA (D227A) fusion proteins

An expression plasmid vector, carrying a kanamycin resistance gene and the lac promoter was constructed for cassette insertion of scFv genes to obtain in frame fusion with a flexible spacer of 18 residues followed by the mutant D227A of the superantigen Staphylococcal Enterotoxin A (SEA) . A Spel site 3' to the phoA promoter and the Xhol site following the scFv were used to excise the signal peptide scFv encoding fragment from the phagemid vector. Single colonies of E. coli UL635 transformed by electroporation with the expression vector containing the inserted scFv-SEA (D227A) genes, were transferred to 96 Micro well plates (Nunc, Denmark) and grown for 17 h at 30°C in 2xYT medium supplemented with 70 μg/ml kanamycin and 2% glucose. Small aliquots (5 μl) were transferred to plates with fresh medium containing antibiotics but without glucose, and cultured at 30°C for 17 h. The Micro well plates were centrifuged and 100 μl of the supernatants were transferred to new plates with an equal volume/well of 1% BSA. Additional induction using IPTG did not increase production of soluble fusion protein in this system (data not shown) . The fusion pro- teins were quantified in a sandwich type ELISA using rabbit anti-SEA antibodies as capture and biotinylated anti-SEA Ig as detector antibodies. The production of the K305 Fab-SEA(D227A) and the recombinant control fusion protein C215 Fab-SEA (D227A) was performed as previously described. EXAMPLES EXAMPLE 1

Immunization of Macaca fascicularis monkeys produced a high serum ti ter against human melanoma cells and HMW-MAA Two Macaca fascicularis monkeys were immunized day 0, 21, 35, and 49 with alun precipitated crude suspensions of human melanoma tumors. The immune sera collected on day 56 were tested for reactivity against melanoma cells and against the high molecular weight melanoma associated antigen (HMW-MAA) . The immune serum of both animals collected day 56 but not the preimmune serum bound to surface antigens on FM3 human melanoma cells in flow cytometry (equally for the two monkeys) . Even at a dilution of 1:40,000 a significant but low shift in flourescence could be detected compared to the preimmune serum at the same dilution (Fig 1A) . A double determinant assay was used to analyse serum reactivity against the human HMW-MAA. The immune serum could be diluted 25,000 times before reactivity reached the background level of the preimmune serum (IB) . No reactivity was found when the melanoma extract or the capture antibody was omitted or when using a capture antibody (mAb C215) against an epithelial antigen not present in the melanoma extract.

HMW-MAA specific scFv antibodies could be identified from a Macaca fascicularis phage library selected against melanoma cells and tissue sections.

The scFv antibody phage library was selected against cryosections of human tissues and melanoma cell lines. The yield of library phage (counted as colony forming units) increased substantially after three repeated selections on cells or tissue sections. The yields were as follows for the three conseqetive rounds: i) FM3 melanoma cells, 6.8xl0^"6, 9.3xl0^"4and l.lxlO^'1 ii) M55M1 melanoma cells, 2.0xl0^"5, 1.5xl0^"4and 5.4xl0^"2 iii) adsorption on spleen tissue sections followed by positive selection on melanoma tissue sections, 1.6xl0^~6, 4.6xl0^"7 and 1.3xl0^"3 iv) adsorption on small bowel sections and positive selection on FM3 melanoma cells, 1.3xl0^~4, 3.5xl0^"6 and 1.3xl0^"3.

Soluble protein from about 200 individual scFv antibody clones were produced from each of the selected lib- raries from the last two selection rounds. These antibodies were screened for binding to melanoma tissue sections and melanoma cell lines using immunohistochemistry and flow cytometry. A secondary antibody directed against the immunological tag, ATPAKSE, was used for the detec- tion in these assays. These screening systems permitted a preliminary evaluation of melanoma reactivity. Positive clones were then recloned in fusion with SEA(D227A) . The latter formate permitted both a sufficiently high production yield in culture supernatants and a more sensitive detection on tissue sections using a rabbit antisera against SEA. Alternatively to the recloning of individual scFv:s, an aliquote of the selected library was recloned into the SEA(D227A) vector for a direct screening in this format. Several scFv-SEA (D227A) antibody-fusion proteins including K305 and K461 (tissue selections on spleen and melanoma) were identified which had an immunohistochemi- cal staining pattern similar to the pattern described for murine antibodies against the HMW-MAA (see below) . All antibodies were identified from the second selection round, demonstrating a lower frequency of binders than the third. EXAMPLE 2

Immunoprecipi taton of a 250 kDa chondroi tinase sensi tive protein by phage selected antibodies FM3 cells were iodinated by the use of a chloramide method as previously described. Briefly, an amount of 50 μg 1, 3 , 4 , 6-tetrachloro-3a-diphenylglycoluril desolved in methylene cloride was airdried in a glass tube. FM3 cells, 13 million in 0.5 ml PBS and 0.5 mCi carrier-free

125I - were incubated in the tube for 10 min on ice with mild agitation. After two washes in PBS the cells were incubated in KI (50 μg/ml) in PBS, for 20 min on ice to reduce unspecific incorporation of 125I - and finally washed again two times in PBS. The iodinated cells were lysed in 2 ml sucrose-NP40 buffer (0.25 M sucrose/50 mM

Tris/1.5 mM MgCl2 /10 mM KCl/l% NP40/protease inhibitor (Boehringer Mannheim, Germany) for 30 min on ice. The lysate was diluted by adding 15 ml PBS and ultracentri- fuged lh at 100 000 g and 4°C. For immunoprecipitation 10 μg primary antibodies were incubated with 1 ml lysate for 1 h followed by 100 μg rabbit-anti -SEA antibodies which were incubated for 20 min and finally with 100 μl Sepharose protein G beads for 20 minutes. The beads were washed two times with 50mM Tris/500 mM NaCl/0.1% NP40. SDS-PAGE using a 4% polyacrylamide gel and protein Western blotting were performed according to standard methods. Autoradiography using film (Amersham) was performed for 1 week at -70°C.

The phage selected antibodies (scFv/Fab -SEA(D227A) fusion proteins) were used for immunoprecipitation using an extract of 125j surface labeled FM3 melanoma cells. The molecular weights of the precipitates were determined by SDS-PAGE. Three antibodies precipitated antigens with a molecular weight which distributed as a smear from a distinct 250 kDa band. Digestion of the precipitate with chondroitinase resulted in a single 250 kDa band with increased intensity. The molecular weight and the sensitivity to chondroitinase support that the antigens recognized are identical to the HMW-MAA precipitated in paral- lell by the murine mAb 9.2.27 (Fig.2). EXAMPLE 3 Epi tope mapping of the selected antibodies

A Nunc Maxisorb plate (Nunc, Denmark) was coated with 35 μl/well lμg/ml mAb 9.2.27 or control antibody o/n 4°C. The incubation fluid was removed and non-specific binding sites were blocked lh at room temperature (RT) in 3% low fat milk (Semper, Sweden) which was removed before incubation for 2 h at RT with the NP-40 extract of melanoma cells diluted 1/10 in 3% milk. The plate was washed 4 times in PBS-0.05% tween 20 (PBST) after this and the following steps. The scFv or Fab-SEA (D227A) fusion proteins at 30 μl/well diluted to lμg/ml or Macaca fascicularis serum diluted 1/50-1/100,000 were incubated 1 h RT. Secondary antibody, 1 μg/ml biotinylated rabbit anti-SEA antibodies or rabbit anti human Ig, was incubated for lh RT and finally extavidin-HRP (Sigma, St. Louis, MO) 1/1000 for 30 min RT. The colour reaction was developed using 3.3 '-diaminobenzidine (Sigma) according to the instructions of the supplier and read at OD405 nm. The binding of selected antibodies was epitope mapped relative to each other and to murine anti-HMW-MAA mAbs. The antibodies, Fab K305 and scFv K461 fused to SEA(D227A) were produced by fermentation and purified by affinity chromatography using immobilized polyclonal rabbit anti-SEA Ig (results not shown) . The yield from 1 liter fermentor cultures of the fusion proteins were 35 mg (K305) and 118 mg (K461) , respectively. HMW-MAA pro- teoglycan in the melanoma extract was captured with mAb 9.2.27. The binding of biotinylated fusion proteins to the captured antigen was competed with using non-biotiny- lated fusion proteins. Alternatively, K305 Fab-SEA (D227A) was used as the capture reagent and murine antibodies were used to inhibit detection with the biotinylated fusion proteins. K305 inhibited K461 (and vice versa) but not another antibody, K458. The murine anti-HMW-MAA mAbs TP41.2 or 763.74 did not inhibit K305 and K461. The K458 antibody was found to be inhibited by the mAb TP41.2 which supports its recognition of an overlapping epitope on the HMW-MAA. Thus, in this assay binding to two different epitopes on the HMW-MAA could be verified, defined by K305/K461 and K458 (Fig.3) . EXAMPLE 4 Affini ty determination on cells and tissue sections of K305 Fab-SEA (D227A)

The K305 Fab-SEA (D227A) fusion protein was iodinated with 2 mCi Na 125I to 80 μg sample according to the iodo- gen method. Cryosections (8mm) were air-dried on multi- well slides, fixed in acetone at -20°C for 10 min and rehydrated in 20% FBS . Iodinated K305 Fab-SEA (D227A) in 20% FBS was added in two-fold dilution series to the sec- tions and incubated lh. The sections were washed four times with TBS and dried before quantifying bound radioactivity of individual wells in a gamma counter. For determination of the affinity using cells, 30 000 FM3 cells/sample were incubated with iodinated K305 Fab- SEA(D227A) at 100 μl/tube in a series of two-fold dilution in 1% BSA for lh and then washed three times in PBS before measuring bound activity. Scatchard plots were constructed using the measured values of bound and added radioactivity. Iodinated antibody K305 Fab-SEA (D227A) was added in dilution series to FM3 cells, or tissue sections of human malignant melanomas and small bowel (to determine smooth muscle reactivity) .

Fig. 4 demonstrates the binding to melanoma tissue. The affinity determined in this experiment was 1.6 nm and the number of binding sites per melanoma tissue section (8 μm thick and approximately 3x4 mm wide) was 4.7xl0⁹. The results from three additional experiments performed on melanoma tissue sections were 0.67, 0.93 and 1.4 nM with 2.2x10^s, 2.9xl0⁹ and 6.4xl0⁹ sites per section. The affinity was also measured on small bowel tissue sections (smooth muscle reactivity) in four experiments, which resulted in 0.82, 0.93, 1.1 and 1.3 nM. In one experiment the cell based system was used giving an affinity of 1.6 nM and 300 000 binding sites per cell. EXAMPLE 5 Tumor and normal tissue reactivi ty of the fusion proteins

All tissues were snap-frozen in isopentane pre- chilled in liquid nitrogen. Cryosections (8mm) were air- dried on slides, fixed in acetone at -20°C for 10 min and rehydrated in 20% foetal bovine serum (FBS) . Endogenous biotin was blocked with avidin for 15 min and then with biotin for 15 min, both of which was deluted 1/6 (Vector Laboratories, Burlingame, CA) . If not otherwise specified, primary antibodies were incubated in a concentration of 5 μg/ml for scFv-SEA (D227A) fusion proteins and lμg/ml for mouse monoclonal antibodies.

Affinity purified and biotinylated rabbit anti-SEA antibodies, 5 μg/ml, and biotinylated rabbit anti -mouse immunoglobulins (DAKO A/S, Denmark), 1/300 of the commercial stock solution, were incubated 30 min followed by StreptABComplex HRP (DAKO A/S, Denmark) , 1/110 of the commercial stock solution, diluted in 50 mM Tris pH 7.6 (TBS) , for 30 min. Between all steps the sections were washed 3 times in TBS. Antibodies, avidin and biotin were all diluted in 20% FBS (fetal bovine serum) in TBS. The staining reaction was developed for 8 min in 0.5 mg/ml

3 , 3 '-diaminobenzidine tetrahydrochloride (Sigma) dissolved in Tris pH 7.6 with 0.01 percent H2O2. After 10 min counterstaining in 0.5 % methyl green, the slides were rinsed for 10 min in tap water and gradually dehydrated in 70-99 % ethanol and xylene before mounting in DPX medium (Sigma) .

The mouse antibodies specific for HMW-MAA, TP41.2 and 763.74, were a kind gift from Dr. Soldano Ferrone and mAb 9.2.27 was obtained from PharMingen, San Diego, CA. The phage selected antibody K305 and the murine anti-HMW-MAA antibody mAb 9.2.27 strongly and homogen- ously stained melanoma cells in seven of eight biopsies of human malignant melanomas surgically removed from different patients. The predominant normal tissue reacti- vity found was a weak staining of smooth muscle, e.g in the gut, and a strong staining of a fraction of blood vessel walls (the smooth muscle layer) in all tissues. Normal tissue reactivity also included a subpopulation basal cells of the epidermis. The K305 antibody showed very weak staining of the uterine smooth muscle cells as compared to mAb 9.2.27. Some blood vessels in the uterus were strongly stained with both antibodies. The murine mAb 9.2.27 also stained cells in the glomerulus, probably mesangial cells, in contrast to K305 (Fig. 5) . Renal blood vessels but not renal tubules were stained by mAb 9.2.27 and K305 (verified by staining of smooth muscle actine and cytokeratin) . None of the phage selected antibodies, K305, and K461 bound to Macaca fascicularis tissues expressing HMW-MAA in contrast to mAb 9.2.27 that demonstrated clear staining of this tissue (not shown) . EXAMPLE 6 K305 Fab-SEA (D227A) mediated superantigen- antibody dependent cellular cytotoxic i ty

Redirected T cell cytotoxicity (superantigen antibody dependent cellular cytotoxicity, SADCC) was measured in a standard 4 h chromium-release assay employing ⁵¹Cr-labeled FM3 or G361 cells as target cells and human T cells as effector cells as previously described. Percent specific lysis was calculated as

100 x com experimental release - cpm background release cpm total release - cpm background release

Two human melanoma target cell lines were used to demonstrate redirection of T cell cytotoxicity to tumor cells by the K305 Fab-SEA (D227A) fusion protein (Fig. 6A) . Dose dependent cellular cytotoxicity could also be achieved with SEA(D227A) alone or fused to irrelevant antibodies (due to a weak interaction with MHC Class II expressed by some melanoma cell lines, e.g. the FM3 line) . However, this cytotoxicity was demonstrated at a hundred-fold higher concentration and with a lower plateau value compared to the antibody targeted K305 Fab- SEA (D227A) cytotoxicity (Fig. 6A) . The targeted cytotoxicity was as expected independent of superantigen presentation on MHC class II, since specific cytotoxicity was also demonstrated for MHC II negative G361 human melanoma cells (Fig. 6B) . EXAMPLE 7

Immuno therapy of established FM3 tumors in SCID mice wi th

K305 Fab-SEA (D227A) g SCID mice were injected I. P. with 3 x 10 FM3 cells in 0.2 ml vehicle (PBS-l%Balb/c mouse serum) . The mice g were injected I. P. with 3 x 10 PBM in 0.2 ml vehicle 4 days later. One to two hours after injection of lymphoid cells all mice were injected I.V. with 100 ug test substance in 0.2 ml vehicle or vehicle alone. Two additional I.V. injections of the test substance were given at three days intervals. The mice were sacrificed by cervical dislocation on day 49 and the number of tumors and the tumor mass was determined. Tumors with a weight of less than 5 mg were estimated as 2 mg, tumors with a weight of more than 5 mg and less than 10 mg as 7 mg and tumors larger than 10 mg with the actual weight. All tumors larger than 1 mg were counted. Each treatment cohort contained five to seven mice to permit comparison with other treatment cohorts treated simultaneously with the same batch of effector cells. Statistical significance was determined by the Mann-Whitney U test using the program Sigma Stat .

The ability of the fusion protein K305 Fab- SEA (D227A) to mediate therapy of FM3 tumors grown intra peritoneally (I. P.) in SCID mice for 4 days was investigated. The therapy was initiated simultaneously to inoculation of human PBM i.p. (humanized SCID model) and compared to vehicle (PBS) and to a control fusion protein, C215 Fab-SEA (D227A) , with an irrelevant Fab moiety. A dramatic and statistically significant reduction of the tumor mass and number was achieved by the K305 Fab- SEA (D227A) fusion protein as compared to the PBS control. Importantly, the effect was significantly higher also compared to the irrelevant fusion protein (Fig. 7), thus demonstrating the dependence of specific antibody-mediated targeting for the therapeutic effect. EXAMPLE 8

Sequencing of antibodies

Briefly, 1.0 pmole/reaction plasmid DNA and 10 pmol/reaction primer DNA were used for automatic sequenc- ing on a PerkinElmer/Applied Biosystem model 373 A. The primers annealed in the lac promoter and in the SEAD(227A) gene, 39 bp upstreem of the Spel and 94 bp downstream of the Sail insertion site of the scFv gene. The annealing temperatures were 60.1°C and 64.9°C, respectively. K305 and K461 scFv were sequenced at

Pharmacia and Upjohn AB, Stockholm, Sweden, using the same sequencing equipment.

The antibodies fall into groups based on the sequence similarity, K305 and K461 belonging to the same group. Comparisons with the closest human germline sequence of the light (lambda) and heavy chain demonstrated more than 89 % sequence identity on the nucleic acid level (Table 1) . All of the antibodies had heavy- chains with close homology to the VH3 family of human germline segments. Human lambda 3 germline family homo- logues were represented by the K305 and K461 antibodies.

The epitope mapping data which suggest that K305 and K461 bind to overlapping epitopes, are supported by the high sequence similarity between the two antibodies (see SEQ ID NO: 2 och SEQ ID NO: 4) . DISCUSSION

The identification and characterization of phage selected primate antibodies directed to two distinct epi- topes on HMW-MAA is disclosed. There is also described the therapeutic use of a selected antibody for super- antigen and T cell dependent growth inhibition of melanoma in humanized SCID mice.

Commonly, tumor associated antigens are defined by murine monoclonal antibodies. However, therapeutic use of murine mAbs frequently leads to the development of human anti-mouse antibodies (HAMA) in patients, which potentially neutralize the effector functions and enhance serum clearance rate. Human monoclonal antibodies should ideally be used, but have by various reasons been diffi- cult to produce by the hybridoma technique. Chimeric antibodies with human constant domains and antibodies with murine CDRs grafted on human Vh/Vl frameworks often demonstrate reduced neutralizing HAMA responses. However responses to V region determinants have been demon- strated.

Another approach is to use non-human primates for the generation of therapeutic antibodies (4) . Primate antibodies should not elicit a response of neutralizing antibodies in man due to their high homology with human. Specific immune responses to human tumor-associated antigens in non-human primates could potentially be less restricted than in man and the reactivity to common species specific tissue antigens should be much reduced as compared to murine responses. The present identification of primate antibodies to human specific (Macaca fascicularis negative) HMW-MAA epitopes including the clone K305 demonstrating a restricted reactivity with uterine smooth muscle and kidney glomeruli as compared to murine mAbs, supports this hypothesis. To be able to identify novel antibodies to epitopes on cell surface expressed tumor- associated antigens, intact cells or suspensions of cells and tissues should preferentially be used as the immu- nogen. A suspension of pooled metastatic melanoma tissue in alun adjuvant were used to immunize the Macaca fasci- cularis monkeys. The immune serum was found to be strongly reactive both with antigens expressed on the surface of human melanoma cells and almost equally strongly and specifically with the human HMW-MAA. This suggests that the HMW-MAA in the melanoma suspension was strongly immu- nodominant to the primate, supported by the finding of high anti-HMW MAA serum anti-body titer in a chimpanzee immunized with purified HMW-MAA.

The scFv antibody phage library was selected on human melanoma tissue sections and melanoma cell lines to generate enriched libraries against both the authentic in vivo phenotype and against cell surface expressed anti- gens. A major advantage of this technology compared to the hybridoma technology is that the identification of novel antibodies and the cloning of their genes is simultaneously performed. This selection of genes encoding tumor reacting antibodies permitted an immediate con- struction of superantigen fusion proteins to be evaluated for tumor targeting properties and therapeutic efficacy. The scFv format was suitable for the selection procedure and for the evaluation of antibody specificities by screening on tissue sections and cell lines. However, due to partial dimer formation of some library selected scFv:s (results not shown) and the lower stability of scFv fragments, Fab-SEA fusion proteins of selected clones were constructed, fermentor cultured and purified. These were needed for detailed immunohistochemical stu- dies, for estimation of true binding affinity and for in vivo experiments.

The K305 Fab-SEA (D227A) fusion protein demonstrated a high binding affinity. The number of binding sites per cell differed less than two-fold from a number of Fab ' s generated from papain cleaved murine mAbs against the

HMW-MAA. Affinity for tissue expressed antigen in melanoma biopsy sections was similar. The number of available binding sites in a tissue section could also be calculated, the result of which could be used for estimation of the in vivo expression level of a therapeutic target. The targeting efficacy of antibodies to solid tumors have

rt o Φ (3 Hi SD rt s. rt ft Ω SD cr rt rt μ- (D (3 rt ft cr rt ft 3 πr SD l-h CD SD πs 0 μ- μ- Φ c m Φ 0 C 3 HI to 0 Φ Φ (D Φ μ-

Φ HI l-h μ- TJ rt μ- rt to g ft Φ X 3 3 rt φ TJ Hi CD Hl

H{ μ- m ^ μ- h{ πr tD 0 Φ πs μ- 0 S3 μ- rt Φ Ω CQ TS 0

SD cr Ω ^yQ cr φ 0 (3 HI CD Hi Ω Hi m CQ 0 πr m 0 Φ μ- Hi

TJ Φ μ- 0 CD H S3 Φ to SD Hi μ- ^ Φ Φ hh Φ ft Hi rt rt

Φ φ Z ^ ft HT rt to rt CD ft Φ rt 0 Ω HI Φ (3 to μ- Φ SD

S3 Φ Hi πr S3 K _. 0 μ- Hi S3 μ- S ^ Hi Φ CD to SD rt m TJ μ- HI HI rt l-h rt 0 Hi hh CD SD SD SD Ω 0 0 ^ HI S3 rt Φ ft CQ πr ft μ- i- _-^■ rr CD Ω Hi rt πr SD Hi μ- m -> CD hh rt 3 Hi φ μ-

Ω μ- ft Φ πr TJ HI CD Ω ft Φ Ω rt m SD ra rt 0 μ- 0 rt SD Hi 0 CQ rt n Φ φ φ 0 rt 0 (D rt Φ • φ Hi cr Hi S3 Hi Φ hh πr Hi

SD μ- Hi rt Hi Ω πs C g rt rt CQ μ- ft πs Φ 3 0 3 rt ft (D

Hi φ μ- C g μ- 1 ft TJ πr πr <! 0 ft rt ft CD 0 μ- 3 ^H HI rt m < g 0 hh πr *< SD φ Φ rt μ- μ- Hi TJ πr ^ μ- Hi CQ 0 Φ CD μ- rr (D 0 Hi μ- S3 HI πr rt Hl 3 SD Hi Φ rt Φ Hi <! μ- cr h-^j rt Hi Φ Ω g ft ^ T SD Φ (D P 0 ft Φ Ω m Φ Φ Φ

0 ^ μ- - μ- SD Φ μ- o Ω O -¹ ft <! Hi Φ • Φ CQ Hi ft 0 Ω rt ns g πs rr Ω O ft SD Φ Φ hh h-¹ TJ CD rt μ- C HI Φ T⁾ μ- 0 CQ Φ Φ (3 0 rt rt Hi m rr μ- H3 h-¹ πr 0

Φ to h-¹ πr Φ TJ πs m CO TJ ω μ- μ- SD SD πs πr SD rt 0 rt

CD Φ 0 SD CO Hi CD rt rt CO Φ φ Φ CQ ft rt Hi φ c <! Φ hh πr

. ft hh to tQ μ- rt SD μ- μ- Hi CO πs CQ μ- 0 CQ ft to μ- φ P φ

Φ TJ g Hj Hi SD cr SD rt 0 Φ rt to Hi rt TJ (D O SD SD tQ μ- πs , CO φ h-¹ πr rt φ h-¹ rr TJ SD

0 0 πs CO rt rt rt Φ rr t CO SD SD μ- TJ rt μ- πr Φ Φ TJ rt ft φ Φ Φ Φ rt rt μ- μ- — - Φ cr < Hi μ- πr Hl Φ hh Ω φ μ- Φ HI CQ CD Φ 0 rt tQ <! φ Φ Φ CQ rt μ- CQ hh μ- S3 ft πs rt φ rt ft ^ Φ Φ <! 0 CQ Ω Φ h rt t φ rt μ- Ω μ- Ω rt μ- Hi S Hi TJ Φ rt 0 TJ SD Ω μ- μ- σ πs CQ rt SD SD πr 0 g 0 0 Hi ft πr hh φ Ω πr TJ rt Ω Ω rt Φ to μ- h-< m SD Hi TJ h CΛ Hi SD φ Φ CD 0 φ SD 0 μ- ι-h S3 o h-¹ rt tQ Hi M Φ rt O SD rr πs m Ω Hi S3 Hi h φ Hi ^ Ω SD 0 rt CD Hi Ω πr 0 Ω μ- TJ Φ φ 0 μ- m <; πr Ti πr μ- rt SD φ HI φ rt rt 3 r CO

Ω CQ 0 (3 g 3 Φ μ- D 0 μ- Hi μ- TJ S SD ^ 0 SD S3

SD rr Φ hh CD T) 3 Hi co t rt CQ CQ 1 SD 1-3 rt rt Hj h-^{1 H}

Hi 0 Ω Φ Hi S3 φ rt to Φ πr X cr C CQ rt Φ Φ rt ft Hi rt rt hh μ- πs πr g -0 πs rt s μ- Ω 3 cr φ 0 Ω • to μ- πr S3 CO Φ SD Φ 0 > rt SD πr S! Φ 0 Φ rt S3 μ- h 0 μ- φ rr Hi ' — ' m Φ 1 μ- Hi πs Φ μ- > S co S3 πs to SD Φ SD Φ Φ rt 2 rt Φ hh ft H⁾ Φ πs Φ

0 CQ CD g m ft TJ Ω SD l-h μ- SD t? SD Hi ft tD rt Hj μ- Hi 0 0 rt Φ c Hi hh cr Hi p> SD CD μ- cr (3 μ- Φ

SD 0 l-h Φ Hi m μ- rt c h-¹ Φ Φ 0 rt rt Hi CO rt to cr rt ^ Ω πs 0 S 0 cr 0 rt φ Ω ft μ- 3 0 3 0 Φ πr 0 0

Φ h-¹ φ rt Ω 0 !*! μ- HI rt CQ _ u___!, Ω hh SD 0 ft cr S h-¹ Hi C h-¹ ft μ- Ω μ- φ 0 Φ Φ 0 μ- Hj Hi H (3 rt "< CO

Hi 0 rt g 0 ^ Ω πs φ Hi TJ πs CD Hi hh SD 0 rt Φ

S3 0 z 0 0 m μ- Φ ft Hi Hi rt 3 μ- Ω ft 3 Hj g rt Φ μ- Hi SD Hi rt hh m φ 3 0 πr SD rr Φ cr Φ φ <

0 φ ft Hi (-^■ Φ ^ hh 0 ft 0 rt SD μ- ft πr ft rt o h-¹ rt ft Φ

Hi μ- φ rt TJ μ- Hi Φ CD rt μ- Φ πr πr Φ Ω

CD i 3 rt Φ μ- rt ft πr μ- ft

3 cr Ω Φ μ- φ SD

^ Φ H SD S 0 1 Φ t

1 1 m SD πs r Ω h-¹ Φ rt CD Φ Φ ft

REFERENCES

1. Pluschke G, Vanek M, Evans A, Dittmar T, Schmid P, Itin P, Filardo EJ, Reisfeld RA (1996) Molecular cloning of a human melanoma-associated chondroitin sulfate proteoglycan. Proc Natl Acad Sci 93:9710

2. Siccardi AG, Burragi GL, Natali PG, Scassellati GA, Viale G, Ferrone S (1990) European multicentre study on melanoma immunoscintigraphy by means of ^^^mTc-labelled monoclonal antibody fragments, the european multicentre group. Eur J Nucl Med 16:317

3. Mittelman A, Chen GZ, Wong GY, Liu C, Hirai S, Ferrone S (1995) Human high moelcular weight-melanoma associated antigen mimicry by mouse anti-idiotypic monoclonal antibody MK2-23: modulation of the immunogenicity in patients with malignant melanoma. Clin Cancer Res 1:705-13

4. Logdberg L, Kaplan E, Drelich M, Harfeldt E, Gunn H, Ehrlich P, Dottavio D, Lake P, Ostberg L (1995) Primate antibodies to components of the human immune system. J Med Primal. 23:285-97

5. Ziai MR, Imberti L, Nicotra MR, Badaracco G, Segatto O, Natali PG, Ferrone S (1987) Analysis with monoclonal antibodies of the molecular and cellular heterogeneity of human high molecular weight melanoma associated antigen. Cancer Res 67:2474

6. Zhao B, Larry R, Helms LR, DesJarlais RL, Abdel- Meduid SS, Wetzel R (1995) A paradigm for drug discovery using a conformation from the crystal structure of a presentation scaffold. Nature Structural Biology, 2:1131- 1137

7. Dougall WC, Petersen NC, Greene MI (1994) Antibody-structure-based design of pharmacological agents. TIBTECH 12:372-379

8. Kovari LC, Momany C, Rossman MG (1995) The use of antibody fragments for crystallization and structure determinations. Structure 3:1291-1293. LEGENDS TO FIGURES

Fig. 1. A) Flow cytometry signal demonstrating strong reactivity against human FM3 melanoma cells of the immune Macaca fascicularis serum collected at day 56 (O) compared to the preimmune serum (•) . B) Specific serum reactivity against the HMW-MAA in a double determinant assay. The HMW-MAA was captured from a NP40 extract of FM3 melanoma cells with the monoclonal antibody 9.2.27 and then detected with the immune serum (□) or the preimmune serum (■) . As further negative controls either the irrelevant murine mAb C215 (Δ) or no antibody (O) was used for capturing or the melanoma extract was omitted (X) .

Fig. 2. Immunoprecipitation of HMW-MAA with phage selected specificities from an 1% NP40 extract of FM3 melanoma cells, surface labeled with 125 _ τ]τ__e molecular weight of the precipitates were determined by SDS-PAGE using a 4 % gel under reducing conditions.

A) Precipitation with the K305 Fab-SEA (D227A) fusion protein. Enzymatic cleavage with chondroitinase (+) eradicated the high molecular weight product and increased the amount of the 250 kDa band. Precipitates using the murine mAb 9.2.27 recognizing HMW-MAA or a murine negative control mAb C2 are shown for comparison. B) The SEA(D227A) fusion proteins K305, K460 and K461 precipitated proteins sensitive to chondroitinase, of identical molecular weight .

Fig. 3. Epitope mapping of scFv/Fab-SEA (D227A) fusion proteins. The HMW-MAA was captured by mAb 9.2.27 from the melanoma extract and detected with biotinylated scFv/Fab-SEA(D227A) fusion proteins. Binding to HMW-MAA of the biotinylated fusion proteins of A) K305 Fab B) K461 scFv and C) K458 scFv inhibited by the non-biotiny- lated fusion proteins K305 Fab (O) , K461 scFv (•) , K458 scFv (A) or with the murine anti-HMW-MAA mAbs TP41.2 (D) or 763.74 (■) . The fusion protein K305 Fab and K461 scFv inhibited each other and K458 scFv was inhibited by the murine mAb TP41.2. Neither of the fusion proteins were inhibited by the mAb 763.74. No specific signal was achieved using the biotinylated negative control C215 Fab-SEA (D227A) reagent (not shown) . Fig. 4. Scatchard plot using tissue sections of metastatic melanoma for the determination of the antibody affinity. The determined affinity of the K305 Fab- SEA(D227A) antibody in this experiment was 1.6 nM and the number of binding sites per section (3x4 mm) was 4.7xl0⁹. Fig. 5. Immunoperoxidase staining of human tissues by the HMW-MAA specific fusion protein K305 Fab- SEA(D227A), in A-E, and by mAb 9.2.27, in F-H. A) Strong and homogenous staining of a malignant melanoma biopsy. B) Staining of the glomerular arteriol (AR) but not with- in the glomerulus in contrast to the staining of glomeru- lus by mAb 9.2.27 in F) . K305 Fab-SEA (D227A) staining of the uterine blood vessels but no/weak staining of the uterine smooth muscle in both high and low concentration, 20 μg/ml in C) , and 0.08μg/ml in D) . MAb 9.2.27 staining of uterine smooth muscle and blood vessels, 0.02 μg/ml in

G) and 0.005 μg/ml in H) . The bar in D) is 100 μm.

Fig. 6. Superantigen antibody dependent cellular cytotoxicity (SADCC) against human melanoma cells . A SEA reactive T cell line established from human PBL stimulat- ed with SEA wild type (12 pM) was added to the melanoma cells in an effector to target ratio of 30:1. a) Lysis of MHCII⁺/HMW-MAA⁺/C215^"/C242^" FM3 melanoma cells with K305 Fab-SEA (D227A) (Δ) was efficient in a 100-fold lower concentration and reached a higher maximum cytotoxicity than SEA(D227A) (X) alone or the irrelevant fusion proteins C215 Fab-SEA (D227A) (•) and C242 Fab- SEA (D227A) (■) . b) The superantigen mediated lysis directed by the antigen specific antibody moiety was MHCII independent, since MHCII^"/HMW-MAA⁺/C215^" G361 human melanoma could be lysed by K305 Fab-SEA (D227A) (Δ) but not by the fusion protein C215 Fab-SEA (D227A) (•) . Fig. 7. Statistically significant growth reduction of established FM3 tumors grown i SCID mice treated with K305 Fab-SEA (D227A) . SCID mice with FM3 tumors grown I.P for 4 days were grafted with human PBM and 2h later treated by I.V. injection of 100 ug of the test substance. The test substance treatment was given three times with three days intervals. The tumor weight and number were cacultated day 49. A: PBM + vehicle (1% Balb/c mouse serum in PBS), B: PBM + K305 Fab-SEA (D227A) , C: PBM + control C215 Fab-SEA (D227A) .

Claims

1. An epitope of high molecular weight melanoma associated antigen (HMW-MAA) , said epitope a) having the ability of being specifically blocked by and to specifically block a binding structure which recognizes said epitope on HMW-MAA, b) being displayed on the surface of human cells but not cells from Macaca fascicularis, and c) having a normal tissue distribution pattern which is more restricted than that of other known epitopes of HMW-MAA.

2. An epitope according to claim 1, wherein the binding structure is labeled and the binding thereof is inhibitable by an unlabeled form of said binding structure and not by other binding structures, and not inhibiting the binding of other binding structures having other specificities.

3. An epitope according to claim 1, wherein said binding structure comprises one or more of the CDR (complementarity determining region) sequences comprising the amino acids number 23-33, 49-55, 88-98 and the amino acids number 158-162, 177-193, 226-240 of the amino acid sequence shown in SEQ ID NO: 2, the amino acids number

22-32, 48-54, 87-97 and the amino acids number 157-161, 176-192, 225-239 of the amino acid sequence shown in SEQ ID NO: 4, or other binding structures having essemtially the same epitope specificity.

4. An epitope according to claim 1, wherein said binding structure is an antibody.

5. An epitope according to claim 4, wherein said antibody comprises the variable region of a light chain comprising essentially the amino acids number 1-109 of the amino acid sequence shown in SEQ ID NO: 2, and the variable region of a heavy chain comprising essentially the amino acids number 128-251 of the amino acid sequence shown in SEQ ID NO : 2.

6. An epitope according to claim 4, wherein said antibody comprises the variable region of a light chain comprising essentially the amino acids number 1-108 of the amino acid sequence shown in SEQ ID NO: 4, and the variable region of a heavy chain comprising essentially the amino acids number 127-250 of the amino acid sequence shown in SEQ ID NO: 4.

7. An epitope according to anyone of claims 1-6, which epitope is displayed less in human uterus and kidneys than other epitopes of the HMW-MAA.

8. An anti-idiotype of an epitope as defined in any one of claims 1-7, which anti-idiotype is specifically blocked by and specifically blocks a binding structure having essemtially the same binding specificity for said epitope.

9. A vaccine composition comprising as an active principle an epitope as defined in anyone of claims 1-7, or an anti-idiotype of said epitope as defined in claim 8.

10. A binding structure which recognizes an epitope of the high molecular weight melanoma associated antigen

(HMW-MAA) and is of organic-chemical nature.

11. A binding structure according to claim 10, which is based on a structure of one or more of the CDR (complementarity determining region) sequences comprising the amino acids number 23-33, 49-55, 88-98 and the amino acids number 158-162, 177-193, 226-240 of the amino acid sequence shown in SEQ ID NO : 2, the amino acids number 22-32, 48-54, 87-97 and the amino acids number 157-161, 176-192, 225-239 of the amino acid sequence shown in SEQ ID NO: 4.

12. An antibody having a binding structure which recognizes a target structure of high molecular weight melanoma associated antigen (HMW-MAA) , and which comprises a) the CDR sequences in the light chain comprising essentially the amino acids number 23-33 (CDR1) , 49-55 (CDR2) and 88-98 (CDR3) of the amino acid sequence shown in SEQ ID No. 2, and the CDR sequences in the heavy chain comprising essentially the amino acids number 158-162 (CDR1) , 177-193 (CDR2) and 226-240 (CDR3) of the amino acid sequence shown in SEQ ID NO: 2, or b) the CDR sequences in the light chain comprising essentially the amino acids number 22-32 (CDR1) , 48-54 (CDR2) , 87-97 (CDR3) of the amino acid sequence shown in SEQ ID NO : 4 , and the CDR sequences in the heavy chain comprising essentially the amino acids number 157-161 (CDR1) , 176-192 (CDR2), 225-239 (CDR3) of the amino acid sequence shown in SEQ ID NO: 4, or other binding structures having essemtially the same epitope specificity.

13. An antibody according to claim 12, which is phage selected.

14. An antibody according to claim 12, wherein the sequences are of Macaca fascicularis origin.

15. An antibody according to claim 12, wherein the sequences have an identity of at least 89% to corresponding sequences of human origin.

16. An antibody according to claim 12, which has low immunogenicity or non- immunogenicity in humans.

17. An antibody according to claim 12, which has been derivatised by genetically linking to other polypeptides, or by chemical conjugation to organic or non-organic chemical molecules, or by oligo- or multimerisation.

18. An antibody according to claim 12, which is genetically linked or chemically conjugated to cytotoxic polypeptides or to cytotoxic organic or non-organic chemical molecules.

19. An antibody according to claim 12, which is genetically linked or chemically conjugated to biologically active molecules.

20. An antibody according to claim 12, which is genetically linked or chemically conjugated to immune activating molecules.

21. An antibody according to claim 12, which has been changed to increase the affinity thereof.

22. An antibody according to claim 12, which has been changed to increase the production yield thereof.

23. An antibody according to claim 12, which has been changed to influence the pharmacokinetic properties thereof.

24. An antibody according to claim 12, which has been changed to give new pharmacokinetic properties thereto.

25. An antibody according to claim 12, which is labeled and the binding thereof is inhibitable by an unlabeled form of said antibody and not by other binding structures, and not inhibiting the binding of other binding structures having other specificities.

26. A pharmaceutical composition comprising as an active principle an antibody as defined in anyone of claims 12-25.

27. A method of in vitro histopathological diagnosis and prognosis of human malignant desease, whereby a sample is contacted with an antibody as defined in anyone of claims 12-25 and an indicator.

28. A method according to claim 27, which method comprises tumor typing.

29. A method according to claim 27, which method comprises tumor screening.

30. A method according to claim 27, which method comprises tumor diagnosis and prognosis.

31. A method according to claim 27, which method comprises monitoring premalignant conditions.

32. A method for in vitro diagnosis and prognosis of human malignant disease, whereby concentrations in bodily fluids of an antigen comprising an epitope, as defined in anyone of claims 1-7, or an anti-idiotype of said epitope, as defined in claim 8, is assayed.

33. A method for in vitro diagnosis and prognosis of human malignant disease, whereby concentrations in bodily fluids of an antibody as defined in anyone of claims 12- 25 is assayed.

34. A method for in vitro diagnosis and prognosis of human malignant disease, whereby concentrations in bodily fluids of a complex of a) an antigen comprising an epi- tope, as defined in anyone of claims 1-7, or an anti- idiotype of said epitope, as defined in claim 8, and b) an antibody, as defined in anyone of claims 12-25, is assayed.

35. A method for in vivo diagnosis and prognosis of human malignant disease, whereby the localisation of an antibody, as defined in anyone of claims 12-25, to tumor deposits in a human subject is determined.

36. A method according to claim 35, whereby said antibody is administered to the subject before the deter- mination.

37. A method according to claim 35, whereby said antibody is accumulated in tumor deposits.

38. A method according to any one of claims 35-37, which is quantitative.

39. A method for therapy of human malignant disease, whereby an antibody as defined in anyone of claims 12-25 is administered to a human subject.

40. A method according to claim 39, whereby said antibody has been changed by being genetically linked to molecules giving the combined molecule changed pharmacokinetic properties.

41. A method according to claim 39, whereby said antibody has been changed by being derivatised.