WO2008119567A2 - Cross-species-specific cd3-epsilon binding domain - Google Patents
Cross-species-specific cd3-epsilon binding domain Download PDFInfo
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- WO2008119567A2 WO2008119567A2 PCT/EP2008/002664 EP2008002664W WO2008119567A2 WO 2008119567 A2 WO2008119567 A2 WO 2008119567A2 EP 2008002664 W EP2008002664 W EP 2008002664W WO 2008119567 A2 WO2008119567 A2 WO 2008119567A2
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Definitions
- the present invention relates to a polypeptide comprising a first human binding domain capable of binding to an epitope of human and non-chimpanzee primate CD3 (epsilon) as well as to a process for the production of the mentioned polypeptide.
- the invention further relates to nucleic acids encoding for the polypeptide, to vectors comprising the same and to host cells comprising the vector.
- the invention provides for a pharmaceutical composition comprising the mentioned polypeptide and medical uses of the polypeptide.
- T cell recognition is mediated by clonotypically distributed alpha beta and gamma delta T cell receptors (TcR) that interact with the peptide-loaded molecules of the peptide MHC (pMHC) (Davis & Bjorkman, Nature 334 (1988), 395-402).
- TcR clonotypically distributed alpha beta and gamma delta T cell receptors
- pMHC peptide MHC
- the antigen- specific chains of the TcR do not possess signalling domains but instead are coupled to the conserved multisubunit signaling apparatus CD3 (Call, Cell 111 (2002), 967- 979, Alarcon, Immunol. Rev. 191 (2003), 38-46, Malissen Immunol. Rev. 191 (2003), 7-27).
- TcR ligation is directly communicated to the signalling apparatus remains a fundamental question in T cell biology (Alarcon, loc. cit.; Davis, Cell 110 (2002), 285-287). It seems clear that sustained T cell responses involve coreceptor engagement, TcR oligomerization, and a higher order arrangement of TcR-pMHC complexes in the immunological synapse (Davis & van der Merwe, Curr. Biol. 11 (2001), R289-R291 , Davis, Nat. Immunol. 4 (2003), 217- 224).
- TcR signalling occurs in the absence of these events and may involve a ligand-induced conformational change in CD3 epsilon (Alarcon, loc. cit, Davis (2002), loc. cit., Gil, J. Biol. Chem. 276 (2001), 11174-11179, Gil, Cell 109 (2002), 901-912).
- the epsilon, gamma, delta and zeta subunits of the signaling complex associate with each other to form a CD3 epsilon-gamma heterodimer, a CD3 epsilon-delta. heterodimer, and a CD3 zeta-zeta homodimer (Call, loc. cit.).
- cysteine-rich stalk appears to play an important role in driving CD3 dimerization (Su, loc. cit., Borroto, J. Biol. Chem. 273 (1998), 12807-12816)
- interaction by means of the extracellular domains of CD3 epsilon and CD3 gamma is sufficient for assembly of these proteins with TcR beta (Manolios, Eur. J. Immunol. 24 (1994), 84-92, Manolios & Li, Immunol. Cell Biol. 73 (1995), 532-536).
- the dominant stoichiometry of the TcR most likely comprises one alpha beta TcR, one CD3 epsilon gamma heterodimer, one CD3 epsilon delta heterodimer and one CD3 zeta zeta homodimer (Call, loc. cit.).
- the crystal structure of this complex bound to the therapeutic antibody OKT3 has recently been elucidated (Kjer-Nielsen, PNAS 101 , (2004), 7675-7680).
- a number of therapeutic strategies modulate T cell immunity by targeting TcR signaling, particularly the anti-human CD3 monoclonal antibodies (mAbs) that are widely used clinically in immunosuppressive regimes.
- the CD3-specific mouse mAb OKT3 was the first mAb licensed for use in humans (Sgro, Toxicology 105 (1995), 23-29) and is widely used clinically as an immunosuppressive agent in transplantation (Chatenoud, Clin. Transplant 7 (1993), 422-430, Chatenoud, Nat. Rev. Immunol. 3 (2003), 123-132, Kumar, Transplant. Proc. 30 (1998), 1351-1352), type 1 diabetes (Chatenoud (2003), loc. cit.), and psoriasis (Utset, J.
- OKT3 has been described in the literature as a potent T cell mitogen (Van Wauve, J. Immunol. 124 (1980), 2708-18) as well as a potent T cell killer (Wong, Transplantation 50 (1990), 683-9). OKT3 exhibits both of these activities in a time-dependent fashion; following early activation of T cells leading to cytokine release, upon further administration OKT3 later blocks all known T cell functions.
- OKT3 has found such wide application as an immunosuppressant in therapy regimens for reduction or even abolition of allograft tissue rejection.
- OKT3 reverses allograft tissue rejection most probably by blocking the function of all T cells, which play a major role in acute rejection.
- OKT3 reacts with and blocks the function of the CD3 complex in the membrane of human T cells, which is associated with the antigen recognition structure of T cells (TCR) and is essential for signal transduction. Which subunit of the TCR/CD3 is bound by OKT3 has been the subject of multiple studies.
- CD3 specific antibodies are listed in Tunnacliffe, Int. Immunol. 1 (1989), 546-50. As indicated above, such CD3 specific antibodies are able to induce various T cell responses such as lymphokine production (Von Wussow, J. Immunol. 127 (1981), 1197; Palacious, J. Immunol. 128 (1982), 337), proliferation (Van Wauve, J. Immunol. 124 (1980), 2708-18) and suppressor-T cell induction (Kunicka, in "Lymphocyte Typing II” 1 (1986), 223). That is, depending on the experimental conditions, CD3 specific monoclonal antibody can either inhibit or induce cytotoxicity (Leewenberg, J. Immunol.
- CD3 antibodies described in the art have been reported to recognize the CD3 epsilon subunit of the CD3 complex, most of them bind in fact to conformational epitopes and, thus, only recognize CD3 epsilon in the native context of the TCR.
- Conformational epitopes are characterized by the presence of two or more discrete amino acid residues which are separated in the primary sequence, but come together on the surface of the molecule when the polypeptide folds into the native protein/antigen (SeIa, (1969) Science 166, 1365 and Laver, (1990) Cell 61, 553-6).
- the conformational epitopes bound by CD3 epsilon antibodies described in the art may be separated in two groups.
- said epitopes are being formed by two CD3 subunits, e.g. of the CD3 epsilon chain and the CD3 gamma or CD3 delta chain.
- CD3 subunits e.g. of the CD3 epsilon chain and the CD3 gamma or CD3 delta chain.
- CD3 epsilon monoclonal antibodies OKT3, WT31 , UCHT1 , 7D6 and Leu-4 did not bind to cells singly transfected with the CD3-epsilon chain.
- these antibodies stained cells doubly transfected with a combination of CD3 epsilon plus either CD3 gamma or CD3 delta (Tunnacliffe, loc. cit.; Law, Int. Immunol.
- the conformational epitope is being formed within the CD3 epsilon subunit itself.
- a member of this group is for instance mAb APA 1/1 which has been raised against denatured CD3 epsilon (Risueno, Blood 106 (2005), 601-8).
- CD3 epsilon antibodies described in the art recognize conformational epitopes located on two or more subunits of CD3.
- the discrete amino acid residues forming the three- dimensional structure of these epitopes may hereby be located either on the CD3 epsilon subunit itself or on the CD3 epsilon subunit and other CD3 subunits such as CD3 gamma or CD3 delta.
- CD3 antibodies have been found to be species-specific.
- Anti-CD3 monoclonal antibodies as holds generally true for any other monoclonal antibodies - function by way of highly specific recognition of their target molecules. They recognize only a single site, or epitope, on their target CD3 molecule.
- OKT-3 one of the most widely used and best characterized monoclonal antibodies specific for the CD3 complex. This antibody reacts with chimpanzee CD3 but not with the CD3 homolog of other primates, such as macaques, or with dog CD3 (Sandusky et al., J. Med. Primatol. 15 (1986), 441-451).
- the anti-CD3 monoclonal antibody UCHT-1 is also reactive with CD3 from chimpanzee but not with CD3 from macaques (own data).
- monoclonal antibodies which recognize macaque antigens, but not their human counterparts.
- monoclonal antibody FN-18 directed to CD3 from macaques (Uda et al., J. Med. Primatol. 30 (2001), 141-147).
- peripheral lymphocytes from about 12% of cynomolgus monkeys lacked reactivity with anti-rhesus monkey CD3 monoclonal antibody (FN-18) due to a polymorphism of the CD3 antigen in macaques.
- Drug candidates can be tested for safety in animals in the following three ways, (i) in a relevant species, i.e. a species where the drug candidates can recognize the ortholog antigens, (ii) in a transgenic animal containing the human antigens and (iii) by use of a surrogate for the drug candidate that can bind the ortholog antigens present in the animal.
- Limitations of transgenic animals are that this technology is typically limited to rodents. Between rodents and man there are significant differences in the physiology and the safety results cannot be easily extrapolated to humans.
- the limitations of a surrogate for the drug candidate are the different composition of matter compared to the actual drug candidate and often the animals used are rodents with the limitation as discussed above.
- preclinical data generated in rodents are of limited predictive power with respect to the drug candidate.
- the approach of choice for safety testing is the use of a relevant species, preferably a lower primate.
- the limitation now of the CD3 binding molecules suitable for therapeutic intervention in man described in the art is that the relevant species are higher primates, in particular chimpanzees. Chimpanzees are considered as endangered species and due to their human-like nature, the use of such animals for drug safety testing has been banned in Europe and is highly restricted elsewhere.
- the present invention relates to a polypeptide comprising a, preferably human, binding domain capable of binding to an epitope of human and non-chimpanzee primate CD3 ⁇ (epsilon) chain, wherein the epitope is part of an amino acid sequence comprised in the group consisting of SEQ ID NOs. 2, 4, 6, or 8. Sequences as shown in SEQ ID NOs. 2, 4, 6 and 8 and functional fragments thereof are context independent CD3 epitopes.
- the advantage of the present invention is the provision of a polypeptide comprising a binding domain exhibiting cross-species specificity to human and non-chimpanzee primate CD3 ⁇ (epsilon) chain, which can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and - in the identical form - as drugs in humans.
- CD3 ⁇ epsilon
- the same molecule can be used in preclinical animal studies as well as in clinical studies in humans. This leads to highly comparable results and a much-increased predictive power of the animal studies compared to species-specific surrogate molecules.
- an N- terminal 1-27 amino acid residue polypeptide fragment of the extracellular domain of CD3 epsilon was surprisingly identified which - in contrast to all other known epitopes of CD3 epsilon described in the art - maintains its three-dimensional structural integrity when taken out of its native environment in the CD3 complex (and fused to a heterologous amino acid sequence such as EpCAM or an immunoglobulin Fc part).
- the context-independence of the CD3 epitope provided in this invention corresponds to the first 27 N-terminal amino acids of CD3 epsilon or functional fragments of this 27 amino acid stretch.
- CD3 epitope in relation to the CD3 epitope means that binding of the herein described inventive binding molecules/antibody molecules does not lead to a change or modification of the conformation, sequence, or structure surrounding the antigenic determinant or epitope.
- CD3 epitope recognized by a conventional CD3 binding molecule (e.g.
- Anti-CD3 binding molecules as part of a bispecific binding molecule as provided herein and generated (and directed) against a context-independent CD3 epitope provide for a surprising clinical improvement with regard to T cell redistribution and, thus, a more favourable safety profile.
- the CD3 binding molecules provided herein induce less allosteric changes in CD3 conformation than the conventional CD3 binding molecules (like molecules provided in WO 99/54440), which recognize context-dependent CD3 epitopes.
- the context-independence of the CD3 epitope of CD3 binding molecules of the invention as part of a bispecific binding molecule is associated with less T cell redistribution during the starting phase of treatment with CD3 binding molecules of the invention resulting in a better safety profile of CD3 binding molecules of the invention compared to conventional CD3 binding molecules known in the art, which recognize context-dependent CD3 epitopes.
- the CD3 binding molecules of the invention by recognizing a context-independent rather than a context-dependent CD3 epitope have a substantial safety advantage over the CD3 binding molecules known in the art.
- CNS adverse events related to T cell redistribution during the starting phase of treatment with conventional CD3 binding molecules usually suffer from confusion and disorientation, in some cases also from urinary incontinence. Confusion is a change in mental status in which the patient is not able to think with his or her usual level of clarity. The patient usually has difficulties to concentrate and thinking is not only blurred and unclear but often significantly slowed down. Patients with CNS adverse events related to T cell redistribution during the starting phase of treatment with conventional CD3 binding molecules may also suffer from loss of memory. Frequently, the confusion leads to the loss of ability to recognize people and/or places, or tell time and the date. Feelings of disorientation are common in confusion, and the decision-making ability is impaired.
- CNS adverse events related to T cell redistribution during the starting phase of treatment with conventional CD3 binding molecules may further comprise blurred speech and/or word finding difficulties. This disorder may impair both, the expression and understanding of language as well as reading and writing. Besides urinary incontinence, also vertigo and dizziness may accompany CNS adverse events related to T cell redistribution during the starting phase of treatment with conventional CD3 binding molecules in some patients.
- the maintenance of the three-dimensional structure within the mentioned 27 amino acid N-terminal polypeptide fragment of CD3 epsilon can be used for the generation of, preferably human, binding domains which are capable of binding to the N-terminal CD3 epsilon polypeptide fragment in vitro and to the native (CD3 epsilon subunit of the) CD3 complex on T cells in vivo with the same binding affinity.
- binding domains which are capable of binding to the N-terminal CD3 epsilon polypeptide fragment in vitro and to the native (CD3 epsilon subunit of the) CD3 complex on T cells in vivo with the same binding affinity.
- CD3 specific antibody molecules as part of a bispecific binding molecule of the invention were used to test for binding to the alanine-mutants of amino acids 1-27 of the N-terminal polypeptide fragment of CD3 epsilon (see appended Example 5).
- the thus isolated, preferably human, binding molecules not only recognize the human N-terminal fragment of CD3 epsilon, but also the corresponding homologous fragments of CD3 epsilon of various primates, including New-World Monkeys (Marmoset, Callithrix jacchus; Saguinus oedipus; Saimiri sciureus) and Old-World Monkeys (Macaca fasciculahs, also known as Cynomolgus Monkey; or Macaca mulatta, also known as Rhesus Monkey).
- New-World Monkeys Marmoset, Callithrix jacchus; Saguinus oedipus; Saimiri sciureus
- Old-World Monkeys Macaca fasciculahs, also known as Cynomolgus Monkey; or Macaca mulatta, also known as Rhesus Monkey.
- said CD3 epsilon specific, preferably human, binding molecules can be integrated into bispecific single chain antibodies in order to generate therapeutics against various diseases, including but not limited to cancer or immunological disorders.
- a surrogate CD3 epsilon binding domain or a bispecific single chain antibody including the same for testing in a phylogenetic distant (from humans) species disappears.
- the very same molecule can be used in animal preclinical testing as is intended to be administered to humans in clinical testing as well as following market approval and therapeutic drug administration.
- the molecule to be used in human therapy in fact differs in sequence and also likely in structure from the surrogate molecule used in preclinical testing in pharmacokinetic parameters and/or biological activity, with the consequence that data obtained in preclinical animal testing have limited applicability / transferability to the human case.
- the use of surrogate molecules requires the construction, production, purification and characterization of a completely new construct. This leads to additional development costs and time necessary to obtain that molecule.
- surrogates have to be developed separately in addition to the actual drug to be used in human therapy, so that two lines of development for two molecules have to be carried out. Therefore, a major advantage of the human binding molecule or a antibody-based constructs exhibiting cross-species specificity described herein is that the identical molecule can be used for therapeutics in humans and in preclinical animal testing.
- binding molecules of the invention capable of binding to an epitope of the human and non-chimpanzee primate CD3 epsilon chain are of human origin.
- the generation of an immune reaction against said binding molecules is excluded to the maximum possible extent upon administration of the binding molecules to human patients.
- CD3 epsilon specific binding molecules as part of a bispecific binding molecule of the invention is their applicability for preclinical testing in various primates.
- the behavior of a drug candidate in animals should ideally be indicative of the expected behavior of this drug candidate upon administration to humans.
- the data obtained from such preclinical testing should therefore generally have a high predictive power for the human case.
- a drug candidate may act differently in a primate species than in humans: Whereas in preclinical testing of said antibody no or only limited adverse effects have been observed in animal studies performed with cynomolgus monkeys, six human patients developed multiple organ failure upon administration of said antibody (Lancet 368 (2006), 2206-7). The results of these not- desired negative events suggest that it may not be sufficient to limit preclinical testing to only one (primate) species.
- the fact that the CD3 epsilon specific human binding molecules of the invention bind to a series of New-World and Old-World Monkeys may help to overcome the problems faced in the case mentioned above. Accordingly, the present invention provides means and methods for minimizing species differences in effects when drugs for human therapy are being developed and tested.
- CD3 epsilon binding domain as part of a bispecific binding molecule of the invention it is also no longer necessary to adapt the test animal to the drug candidate intended for administration to humans, such as e.g. the creation of transgenic animals.
- the CD3 epsilon specific, preferably human, binding molecules (or bispecific single chain antibodies containing the same), exhibiting cross-species specificity according to the uses and the methods of invention can be directly used for preclinical testing in non-chimpanzee primates, without any genetic manipulation of the animals.
- a further advantage of the uses of the CD3 epsilon specific, preferably human, binding molecules (or bispecific single chain antibodies containing the same) exhibiting cross-species specificity is the fact that chimpanzees as an endangered species are avoided for animal testing.
- Chimpanzees are the closest relatives to humans and were recently grouped into the family of hominids based on the genome sequencing data (Wildman et al., PNAS 100 (2003), 7181). Therefore, data obtained with chimpanzee is generally considered to be highly predictive for humans.
- the number of chimpanzees, which can be used for medical experiments is highly restricted. As stated above, maintenance of chimpanzees for animal testing is therefore both costly and ethically problematic.
- CD3 epsilon specific, preferably human, binding molecules of the invention avoids both ethical objections and financial burden during preclinical testing without prejudicing the quality, i.e. applicability, of the animal testing data obtained.
- the uses of CD3 epsilon specific, preferably human, binding molecules (or bispecific single chain antibodies containing the same) provides for a reasonable alternative for studies in chimpanzees.
- CD3 epsilon specific, preferably human, binding molecules of the invention is the ability of extracting multiple blood samples when using it as part of animal preclinical testing, for example in the course of pharmacokinetic animal studies. Multiple blood extractions can be much more readily obtained with a non-chimpanzee primate than with lower animals, e.g. a mouse. The extraction of multiple blood samples allows continuous testing of blood parameters for the determination of the biological effects induced by the, preferably human, binding molecule (or bispecific single chain antibody) of the invention.
- the extraction of multiple blood samples enables the researcher to evaluate the pharmacokinetic profile of the, preferably human, binding molecule (or bispecific single chain antibody containing the same) as defined herein.
- potential side effects which may be induced by said, preferably human, binding molecule (or bispecific single chain antibody containing the same) reflected in blood parameters can be measured in different blood samples extracted during the course of the administration of said antibody. This allows the determination of the potential toxicity profile of the, preferably human, binding molecule (or bispecific single chain antibody containing the same) as defined herein.
- the advantages of the, preferably human, binding molecules (or bispecific single chain antibodies) as defined herein exhibiting cross-species specificity may be briefly summarized as follows:
- the, preferably human, binding molecules (or bispecific single chain antibodies) as defined herein used in preclinical testing is the same as the one used in human therapy.
- the uses of the, preferably human, binding molecules (or bispecific single chain antibodies) as defined herein for the preparation of therapeutics in human is less cost- and labor-intensive than surrogate approaches.
- the, preferably human, binding molecules (or bispecific single chain antibodies) as defined herein can be used for preclinical testing not only in one primate species, but in a series of different primate species, thereby limiting the risk of potential species differences between primates and human.
- chimpanzee as an endangered species for animal testing is avoided.
- multiple blood samples can be extracted for extensive pharmacokinetic studies.
- the generation of an immune reaction against said binding molecules is minimalized when administered to human patients. Induction of an immune response with antibodies specific for a drug candidate derived from a non-human species as e.g. a mouse leading to the development of human-anti-mouse antibodies (HAMAs) against therapeutic molecules of murine origin is excluded.
- HAMAs human-anti-mouse antibodies
- protein is well known in the art and describes biological compounds. Proteins comprise one or more amino acid chains (polypeptides), whereby the amino acids are bound among one another via a peptide bond.
- polypeptide as used herein describes a group of molecules, which consist of more than 30 amino acids.
- the group of polypeptides comprises “proteins” as long as the proteins consist of a single polypeptide. Also in line with the definition the term “polypeptide” describes fragments of proteins as long as these fragments consist of more than 30 amino acids. Polypeptides may further form multimers such as dimers, trimers and higher oligomers, i.e. consisting of more than one polypeptide molecule.
- Polypeptide molecules forming such dimers, trimers etc. may be identical or non-identical.
- the corresponding higher order structures of such multimers are, consequently, termed homo- or heterodimers, homo- or heterotrimers etc.
- An example for a hereteromultimer is an antibody molecule, which, in its naturally occurring form, consists of two identical light polypeptide chains and two identical heavy polypeptide chains.
- the terms "polypeptide” and "protein” also refer to naturally modified polypeptides/proteins wherein the modification is effected e.g. by post-translational modifications like glycosylation, acetylation, phosphorylation and the like. Such modifications are well known in the art.
- human and “man” refers to the species Homo sapiens. As far as the medical uses of the constructs described herein are concerned, human patients are to be treated with the same molecule.
- human origin as used in the context with the molecules of the invention describes molecules derivable from human libraries or having a structure/sequence corresponding to the human equivalent. Accordingly, proteins having an amino acid sequence corresponding to the analog human sequence, e.g. an antibody fragment having an amino acid sequences in the framework corresponding to the human germline sequences, are understood as molecules of human origin.
- a “non-chimpanzee primate” or “non-chimp primate” or grammatical variants thereof refers to any primate other than chimpanzee, i.e. other than an animal of belonging to the genus Pan, and including the species Pan paniscus and Pan troglodytes, also known as Anthropopithecus troglodytes or Simia satyrus.
- a “primate”, “primate species”, “primates” or grammatical variants thereof denote/s an order of eutherian mammals divided into the two suborders of prosimians and anthropoids and comprising man, apes, monkeys and lemurs.
- primates as used herein comprises the suborder Strepsirrhini (non-tarsier prosimians), including the infraorder Lemuhformes (itself including the superfamilies Cheirogaleoidea and Lemuroidea), the infraorder Chiromyiformes (itself including the family Daubentoniidae) and the infraorder Lohsiformes (itself including the families Lohsidae and Galagidae).
- Strepsirrhini non-tarsier prosimians
- the infraorder Lemuhformes itself including the superfamilies Cheirogaleoidea and Lemuroidea
- Chiromyiformes itself including the family Daubentoniidae
- Lohsiformes itself including the families Lohsidae and Galagidae
- Principals as used herein also comprises the suborder Haplorrhini, including the infraorder Tarsiiformes (itself including the family Tarsiidae), the infraorder Simiiformes (itself including the Platyrrhini, or New-World monkeys, and the Catarrhini, including the Cercopithecidea, or Old-World Monkeys).
- non-chimpanzee primate species may be understood within the meaning of the invention to be a lemur, a tarsier, a gibbon, a marmoset (belonging to New-World Monkeys of the family Cebidae) or an Old-World Monkey (belonging to the superfamily Cercopithecoidea).
- an "Old-World Monkey” comprises any monkey falling in the superfamily Cercopithecoidea, itself subdivided into the families: the Cercopithecinae, which are mainly African but include the diverse genus of macaques which are Asian and North African; and the Colobinae, which include most of the Asian genera but also the African colobus monkeys.
- an advantageous non-chimpanzee primate may be from the Tribe Cercopithecini, within the genus Allenopithecus (Allen's Swamp Monkey, Allenopithecus nigroviridis); within the genus Miopithecus (Angolan Talapoin, Miopithecus talapoin; Gabon Talapoin, Miopithecus ogouensis); within the genus Erythrocebus (Patas Monkey, Erythrocebus patas); within the genus Chlorocebus (Green Monkey, Chlorocebus sabaceus; Grivet, Chlorocebus aethiops; Bale Mountains Vervet, Chlorocebus djamdjamensis; Tantalus Monkey, Chlorocebus tantalus; Vervet Monkey, Chlorocebus pygerythrus; Malbrouck, Chlorocebus cynosuros
- an advantageous non-chimpanzee primate also within the subfamily Cercopithecinae but within the Tribe Papionini, may be from within the genus Macaca (Barbary Macaque, Macaca sylvanus; Lion-tailed Macaque, Macaca silenus; Southern Pig-tailed Macaque or Beruk, Macaca nemestrina; Northern Pig-tailed Macaque, Macaca leonina; Pagai Island Macaque or Bokkoi, Macaca pagensis; Siberut Macaque, Macaca siberu; Moor Macaque, Macaca maura; Booted Macaque, Macaca ochreata; Tonkean Macaque, Macaca tonkeana; Heck's Macaque, Macaca homei; Gorontalo Macaque, Macaca nigriscens; Celebes Crested Macaque or Black "Ape", Macaca nigra; Cyno
- Macaca fascic ⁇ la ⁇ s also known as Cynomolgus monkey and, therefore, in the Examples named “Cynomolgus”
- Macaca mulatta rhesus monkey, named “rhesus”
- an advantageous non-chimpanzee primate may be from the African group, within the genus Colobus (Black Colobus, Colobus satanas; Angola Colobus, Colobus angolensis; King Colobus, Colobus polykomos; Ursine Colobus, Colobus vellerosus; Mantled Guereza, Colobus guereza); within the genus Piliocolobus (Western Red Colobus, Piliocolobus badius; Piliocolobus badius badius; Piliocolobus badius temminckii; Piliocolobus badius waldronae; Pennant's Colobus, Piliocolobus pennantii; Piliocolobus pennantii pennantii; Piliocolobus pennantii epieni; Piliocolobus pennantii bouvieri; Preuss's Red Colobus, Piliocolobus pre
- an advantageous non-chimpanzee primate may alternatively be from the Langur (leaf monkey) group, within the genus Semnopithecus (Nepal Gray Langur, Semnopithecus schistaceus; Kashmir Gray Langur, Semnopithecus ajax; Tarai Gray Langur, Semnopithecus hector, Northern Plains Gray Langur, Semnopithecus entellus; Black-footed Gray Langur, Semnopithecus hypoleucos; Southern Plains Gray Langur, Semnopithecus dussumie ⁇ ; Tufted Gray Langur, Semnopithecus priam); within the T.
- obscurus group of the genus Trachypithecus (Dusky Leaf Monkey or Spectacled Leaf Monkey, Trachypithecus obscurus; Phayre's Leaf Monkey, Trachypithecus phayrei); within the T. pileatus group of the genus Trachypithecus (Capped Langur, Trachypithecus pileatus; Shortridge's Langur, Trachypithecus shortridgei; Gee's Golden Langur, Trachypithecus geei); within the T.
- an advantageous non-chimpanzee primate may alternatively be from the Odd-Nosed group, within the genus Pygathrix (Red-shanked Douc, Pygathrix nemaeus; Black-shanked Douc, Pygathrix nigripes; Gray-shanked Douc, Pygathrix cinema); within the genus Rhinopithecus (Golden Snub-nosed Monkey, Rhinopithecus roxellana; Black Snub-nosed Monkey, Rhinopithecus bieti; Gray Snub-nosed Monkey, Rhinopithecus brelichi; Tonkin Snub-nosed Langur, Rhinopithecus avunculus); within the genus Nasalis (Proboscis Monkey, Nasalis larvatus); or within the genus Simias (Pig-tailed Langur, Simias concolor).
- the term "marmoset” denotes any New-World Monkeys of the genus Callithrix, for example belonging to the Atlantic marmosets of subgenus Callithrix (sic!) (Common Marmoset, Callithrix (Callithrix) jacchus; Black-tufted Marmoset, Callithrix (Callithrix) penicillata; Wied's Marmoset, Callithrix (Callithrix) kuhlii; White- headed Marmoset, Callithrix (Callithrix) geoffroyi; Buffy-headed Marmoset, Callithrix (Callith ⁇ x) flaviceps; Buffy-tufted Marmoset, Callithrix (Callithrix) aurita); belonging to the Amazonian marmosets of subgenus Mico (Rio Acari Marmoset, Callithrix (Mico) acariensis; Manicore Marmoset, Callithrix (M
- Other genera of the New-World Monkeys comprise tamarins of the genus Saguinus (comprising the S. oed/pus-group, the S. midas group, the S. nigricollis group, the S. mystax group, the S. bicolor group and the S. inustus group) and squirrel monkeys of the genus Samiri (e.g. Saimiri sciureus, Saimiri oerstedii, Saimiri ustus, Saimiri boliviensis, Saimiri vanzolini)
- Samiri e.g. Saimiri sciureus, Saimiri oerstedii, Saimiri ustus, Saimiri boliviensis, Saimiri vanzolini
- binding domain characterizes in connection with the present invention a domain of a polypeptide which specifically binds/interacts with a given target structure/antigen/epitope.
- the binding domain is an "antigen-interaction-site".
- antiigen-interaction-site defines, in accordance with the present invention, a motif of a polypeptide, which is able to specifically interact with a specific antigen or a specific group of antigens, e.g. the identical antigen in different species. Said binding/interaction is also understood to define a "specific recognition”.
- the term "specifically recognizing” means in accordance with this invention that the antibody molecule is capable of specifically interacting with and/or binding to at least two, preferably at least three, more preferably at least four amino acids of an antigen, e.g. the human CD3 antigen as defined herein.
- an antigen e.g. the human CD3 antigen as defined herein.
- Such binding may be exemplified by the specificity of a "lock-and-key-principle".
- specific motifs in the amino acid sequence of the binding domain and the antigen bind to each other as a result of their primary, secondary or tertiary structure as well as the result of secondary modifications of said structure.
- the specific interaction of the antigen-interaction-site with its specific antigen may result as well in a simple binding of said site to the antigen.
- a binding domain in line with the present invention is an antibody.
- the binding domain may be a monoclonal or polyclonal antibody or derived from a monoclonal or polyclonal antibody.
- antibody comprises derivatives or functional fragments thereof which still retain the binding specificity. Techniques for the production of antibodies are well known in the art and described, e.g.
- antibody also comprises immunoglobulins (Ig's) of different classes (i.e. IgA, IgG, IgM, IgD and IgE) and subclasses (such as IgGI , lgG2 etc.).
- Ig's immunoglobulins of different classes (i.e. IgA, IgG, IgM, IgD and IgE) and subclasses (such as IgGI , lgG2 etc.).
- antibodies can be used, for example, for the immunoprecipitation, affinity purification and immunolocalization of the polypeptides or fusion proteins of the invention as well as for the monitoring of the presence and amount of such polypeptides, for example, in cultures of recombinant prokaryotes or eukaryotic cells or organisms.
- antibody also includes embodiments such as chimeric, single chain and humanized antibodies, as well as antibody fragments, like, inter alia, Fab fragments.
- Antibody fragments or derivatives further comprise F(ab')2, Fv, scFv fragments or single domain antibodies, single variable domain antibodies or immunoglobulin single variable domain comprising merely one variable domain, which might be VH or VL, that specifically bind an antigen or epitope independently of other V regions or domains; see, for example, Harlow and Lane (1988) and (1999), loc. cit.
- immunoglobulin single variable domain encompasses not only an isolated antibody single variable domain polypeptide, but also larger polypeptides that comprise one or more monomers of an antibody single variable domain polypeptide sequence.
- the (antibody) derivatives can be produced by peptidomimetics.
- techniques described for the production of single chain antibodies see, inter alia, US Patent 4,946,778) can be adapted to produce single chain antibodies specific for elected polypeptide(s).
- transgenic animals may be used to express humanized antibodies specific for polypeptides and fusion proteins of this invention.
- any technique, providing antibodies produced by continuous cell line cultures can be used.
- Examples for such techniques include the hybridoma technique (K ⁇ hler and Milstein Nature 256 (1975), 495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor, Immunology Today 4 (1983), 72) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96).
- antibody comprises antibody constructs, which may be expressed in a host as described herein below, e.g. antibody constructs which may be transfected and/or transduced via, inter alia, viruses or plasmid vectors.
- binding (domain) molecule does not or does not significantly cross- react with polypeptides which have similar structure as those bound by the binding molecule, and which might be expressed by the same cells as the polypeptide of interest.
- Cross-reactivity of a panel of binding molecules under investigation may be tested, for example, by assessing binding of said panel of binding molecules under conventional conditions (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988 and Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999).
- Examples for the specific interaction of a binding domain with a specific antigen comprise the specificity of a ligand for its receptor.
- Said definition particularly comprises the interaction of ligands, which induce a signal upon binding to its specific receptor.
- Examples for said interaction is the interaction of an antigenic determinant (epitope) with the binding domain (antigenic binding site) of an antibody.
- cross-species specificity means binding of a binding domain described herein to the same target molecule in humans and non-chimpanzee primates.
- cross-species specificity or “interspecies specificity” is to be understood as an interspecies reactivity to the same molecule X expressed in different species, but not to a molecule other than X.
- Cross- species specificity of a monoclonal antibody recognizing e.g. human CD3 epsilon, to a non-chimpanzee primate CD3 epsilon, e.g. macaque CD3 epsilon can be determined, for instance, by FACS analysis.
- the FACS analysis is carried out in a way that the respective monoclonal antibody is tested for binding to human and non- chimpanzee primate cells, e.g. macaque cells, expressing said human and non- chimpanzee primate CD3 epsilon antigens, respectively.
- An appropriate assay is shown in the following examples.
- CD3 epsilon denotes a molecule expressed as part of the T cell receptor and has the meaning as typically ascribed to it in the prior art. In human, it encompasses in individual or independently combined form all known CD3 subunits, for example CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha and CD3 beta.
- the non-chimpanzee primate CD3 antigens as referred to herein are, for example, Macaca fascicula ⁇ s CD3 and Macaca mulatta CD3.
- Macaca fascicularis it encompasses CD3 epsilon FN-18 negative and CD3 epsilon FN-18 positive, CD3 gamma and CD3 delta.
- Macaca mulatta it encompasses CD3 epsilon, CD3 gamma and CD3 delta.
- said CD3 as used herein is CD3 epsilon.
- the human CD3 epsilon is indicated in GenBank Accession No.NM_000733 and comprises SEQ ID NO. 1.
- the human CD3 gamma is indicated in GenBank Accession NO. NM_000073.
- the human CD3 delta is indicated in GenBank Accession No. NM_000732.
- CD3 epsilon "FN-18 negative" of Macaca fascicularis i.e. CD3 epsilon not recognized by monoclonal antibody FN-18 due to a polymorphism as set forth above
- GenBank Accession No. AB073994 The CD3 epsilon "FN-18 negative" of Macaca fascicularis (i.e. CD3 epsilon not recognized by monoclonal antibody FN-18 due to a polymorphism as set forth above) is indicated in GenBank Accession No. AB073994.
- the CD3 epsilon "FN-18 positive" of Macaca fascicularis i.e. CD3 epsilon recognized by monoclonal antibody FN-18
- GenBank Accession No. AB073993 The CD3 gamma of Macaca fascicularis is indicated in GenBank Accession No. AB073992.
- the CD3 delta of Macaca fascicularis is indicated in GenBank Accession No. AB073991.
- the nucleic acid sequences and amino acid sequences of the respective CD3 epsilon, gamma and delta homologs of Macaca mulatta can be identified and isolated by recombinant techniques described in the art (Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 3 rd edition 2001). This applies mutatis mutandis to the CD3 epsilon, gamma and delta homologs of other non-chimpanzee primates as defined herein.
- the identification of the amino acid sequence of Callithrix jacchus, Saimiri sciureus und Saguinus oedipus is described in the appended examples.
- the amino acid sequence of the extracellular domain of the CD3 epsilon of Callithrix jacchus is depicted in SEQ ID NO: 3, the one of Saguinus oedipus is depicted in SEQ ID NO: 5 and the one of Saimiri sciureus is depicted in SEQ ID NO: 7.
- epitope defines an antigenic determinant, which is specifically bound/identified by a binding molecule as defined above.
- the binding domain or molecules may specifically bind to/interact with conformational or continuous epitopes, which are unique for the target structure, e.g. the human and non-chimpanzee primate CD3 epsilon chain.
- a conformational or discontinuous epitope is characterized for polypeptide antigens by the presence of two or more discrete amino acid residues which are separated in the primary sequence, but come together on the surface of the molecule when the polypeptide folds into the native protein/antigen (SeIa, (1969) Science 166, 1365 and Laver, (1990) Cell 61 , 553-6).
- the two or more discrete amino acid residues contributing to the epitope are present on separate sections of one or more polypeptide chain(s). These residues come together on the surface of the molecule when the polypeptide chain(s) fold(s) into a three-dimensional structure to constitute the epitope.
- a continuous or linear epitope consists of two or more discrete amino acid residues, which are present in a single linear segment of a polypeptide chain.
- a "context-dependent" CD3 epitope refers to the conformation of said epitope.
- a context-independent CD3 epitope refers to an N-terminal 1-27 amino acid residue polypeptide or a functional fragment thereof of CD3 epsilon. This N-terminal 1-27 amino acid residue polypeptide or a functional fragment thereof maintains its three- dimensional structural integrity and correct conformation when taken out of its native environment in the CD3 complex.
- the context-independency of the N-terminal 1-27 amino acid residue polypeptide or a functional fragment thereof, which is part of the extracellular domain of CD3 epsilon, represents, thus, an epitope which is completely different to the epitopes of CD3 epsilon described in connection with a method for the preparation of human binding molecules in WO 2004/106380. Said method used solely expressed recombinant CD3 epsilon.
- binding molecules as disclosed in WO 2004/106380 are not capable of binding to the N-terminal 1-27 amino acid residues of the CD3 epsilon chain.
- conventional anti-CD3 binding molecules or anti-CD3 antibody molecules bind CD3 epsilon chain at a position which is more C-terminally located than the context-independent N-terminal 1-27 amino acid residue polypeptide or a functional fragment provided herein.
- Prior art antibody molecules OKT3 and UCHT-1 have also a specificity for the epsilon-subunit of the TCR/CD3 complex between amino acid residues 35 to 85 and, accordingly, the epitope of these antibodies is also more C-terminally located.
- UCHT-1 binds to the CD3 epsilon chain in a region between amino acid residues 43 to 77 (Tunnacliffe, Int. Immunol. 1 (1989), 546-50; Kjer-Nielsen, PNAS 101 , (2004), 7675- 7680; Salmeron, J. Immunol. 147 (1991), 3047-52). Therefore, prior art anti-CD3 molecules do not bind to and are not directed against the herein defined context- independent N-terminal 1-27 amino acid residue epitope (or a functional fragment thereof).
- binding domain comprised in a polypeptide of the invention, e.g. in a bispecific single chain antibody as defined herein, e.g. monoclonal antibodies binding to both the human and non-chimpanzee primate CD3 epsilon (e.g. macaque CD3 epsilon) can be used.
- a bispecific single chain antibody as defined herein, e.g. monoclonal antibodies binding to both the human and non-chimpanzee primate CD3 epsilon (e.g. macaque CD3 epsilon) can be used.
- the non-chimpanzee primate is an old world monkey.
- the old world monkey is a monkey of the Papio genus Macaque genus.
- the monkey of the Macaque genus is Assamese macaque (Macaca assamensis), Barbary macaque (Macaca sylvanus), Bonnet macaque (Macaca radiata), Booted or Sulawesi-Booted macaque (Macaca ochreata), Sulawesi-crested macaque (Macaca nigra), Formosan rock macaque (Macaca cyclopsis), Japanese snow macaque or Japanese macaque (Macaca fuscata), Cynomologus monkey or crab-eating macaque or long-tailed macaque or Java macaque (Macaca fascicularis), Lion-tailed macaque (Macaca silenus), Pigtailed macaque (Macaca nemest ⁇ na), Rhesus macaque (Macaca mulatta), Vietnamese macaque (Macaca thibetana), Tonkean macaque (Macaca tonke
- the monkey of the Papio genus is Hamadryas Baboon, Papio hamadryas; Guinea Baboon, Papio papio; Olive Baboon, Papio anubis; Yellow Baboon, Papio cynocephalus; Chacma Baboon, Papio ursinus
- the non- chimpanzee primate is a new world monkey.
- the new world monkey is a monkey of the Callithrix genus (marmoset), the Saguinus genus or the Samiri genus.
- the monkey of the Callithrix genus is Callithrix jacchus
- the monkey of the Saguinus genus is Saguinus oedipus
- the monkey of the Samiri genus is Saimiri sciureus.
- polypeptide of the invention binds with the first binding domain to an epitope of human and non-chimpanzee primate CD3 ⁇ (epsilon) chain, wherein the epitope is part of an amino acid sequence comprised in the group consisting of 27 amino acid residues as depicted in SEQ ID NOs. 2, 4, 6, or 8 or a functional fragment thereof.
- said epitope is part of an amino acid sequence comprising 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6 or 5 amino acids. More preferably, wherein said epitope comprises at least the amino acid sequence Gln-Asp-Gly-Asn-Glu (Q-D-G-N-E-D).
- a functional fragment of the N-terminal 1-27 amino acid residues means that said functional fragment is still a context-independent epitope maintaining its three-dimensional structural integrity when taken out of its native environment in the CD3 complex (and fused to a heterologous amino acid sequence such as EpCAM or an immunoglobulin Fc part, e.g. as shown in Example 3.1).
- the maintenance of the three-dimensional structure within the 27 amino acid N-terminal polypeptide or functional fragment thereof of CD3 epsilon can be used for the generation of binding domains which bind to the N-terminal CD3 epsilon polypeptide fragment in vitro and to the native (CD3 epsilon subunit of the) CD3 complex on T cells in vivo with the same binding affinity.
- a functional fragment of the N-terminal 1-27 amino acid residues means that CD3 binding molecules provided herein can still bind to such functional fragments in a context- independent manner.
- the person skilled in the art is aware of methods for epitope mapping to determine which amino acid residues of an epitope are recognized by such anti-CD3 binding molecules (e.g. alanine scanning).
- the polypeptide of the invention comprises a (first) binding domain capable of binding to an epitope of human and non-chimpanzee primate CD3 ⁇ chain and a second binding domain capable of binding to a cell surface antigen.
- the term "cell surface antigen” as used herein denotes a molecule, which is displayed on the surface of a cell. In most cases, this molecule will be located in or on the plasma membrane of the cell such that at least part of this molecule remains accessible from outside the cell in tertiary form.
- a non-limiting example of a cell surface molecule, which is located in the plasma membrane is a transmembrane protein comprising, in its tertiary conformation, regions of hydrophilicity and hydrophobicity.
- At least one hydrophobic region allows the cell surface molecule to be embedded, or inserted in the hydrophobic plasma membrane of the cell while the hydrophilic regions extend on either side of the plasma membrane into the cytoplasm and extracellular space, respectively.
- cell surface molecules which are located on the plasma membrane are proteins which have been modified at a cysteine residue to bear a palmitoyl group, proteins modified at a C-terminal cysteine residue to bear a farnesyl group or proteins which have been modified at the C-terminus to bear a glycosyl phosphatidyl inositol (“GPI”) anchor. These groups allow covalent attachment of proteins to the outer surface of the plasma membrane, where they remain accessible for recognition by extracellular molecules such as antibodies.
- GPI glycosyl phosphatidyl inositol
- cell surface antigens examples include EGFR, EGFRvIII, MCSP, Carbonic anhydrase IX (CAIX), CD30, CD33, Her2/neu, IgE, CD44v6 and Muc-1.
- examples for corresponding cell surface antibodies comprise antigens which are characteristic for a specific disease or ailment, i.e. cancer, autoimmune diseases or infections diseases including viral infections. Accordingly, the term "cell surface antigens" explicitly includes viral proteins such as native, unprocessed viral proteins exposed on the surface of infected cells (described inter alia for envelope proteins of Hepatitis virus B, C and HIV-1).
- cytotoxic T cells One defense function of cytotoxic T cells is the destruction of virus-infected cells, therefore, the unique property of the bispecific binding molecules of the invention to activate and redirect cytotoxic T cells irrespecitve of their autochthonous specificity has a great impact on the broad field of chronic virus infections. For the majority of these infections elimination of persistently infected cells is the only chance for cure.
- Adoptive T cell therapies are currently being developed against chronic CMV and EBV infections (Rooney, CM., et al., Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr-virus-related lymphoproliferation. Lancet, 1995. 345 (8941): p.
- Chronic hepatitis B infection is clearly one of the most interesting and rewarding indications.
- Current treatment of chronic HBV hepatitis rests on interferon gamma and nucleosid or nucleotide analogues, a long term therapy with considerable side-effects such as induction of hepatitis flares, fever, myalgias, thrombocytopenia and depression.
- a persistent inflammation in chronic hepatitis B leads to liver cirrhosis and hepatocellular carcinoma in more than 25% of patients.
- up to 40 % of patients with chronic hepatitis B will die from serious complications, accounting for 0.6 to 1.0 million deaths per year worldwide
- HBV the prototype of the Hepadnaviruses is an enveloped virus whose relaxed circular (re) genome is reverse transcribed into an RNA pregenome. After infection the re DNA is imported into the hepatocyte nucleus where it is completed to a covalently closed circular DNA (cccDNA) containing four overlapping reading frames. It serves as transcription template for the pregenomic RNA and three subgenomic RNAs.
- the RNA pregenome functions as mRNA for translation of the viral core and polymerase protein.
- Infected cells produce continuously HBV surface protein (HBsAg) from the cccDNA even when HBV replication is stopped.
- HBsAg consists of the small surface (S) proteins with very few portions of middle and large (L) surface proteins.
- HBV S and L are targeted to the Endoplasmatic Reticulum (ER) membrane from where they are transported in membrane vesicles via the trans golgi organelle to the plasma membrane (Gorelick, F. S. and C Shugrue, Exiting the endoplasmic reticulum. MoI Cell Endocrinol, 2001. 177 (1-2): p. 13-8).
- S and L proteins are permanently expressed on the surface of HBV replicating hepatocytes as shown recently (Chu, CM. and Y.F. Liaw, Membrane staining for hepatitis B surface antigen on hepatocytes: a sensitive and specific marker of active viral replication in hepatitis B. J Clin Pathol, 1995.
- HBV Hepatitis virus B
- HCV Hepatitis virus C
- HIV-1 HIV-1
- T cells modified by a chimeric TCR with an Fv antibody construct directed at the gp120 envelope protein can kill HIV-1 infected target cells (Masiero, S., et al., T-cell engineering by a chimeric T-cell receptor with antibody-type specificity for the HIV-1 gp120. Gene Ther, 2005. 12 (4): p. 299-310).
- hepatitis virus B expresses the envelope protein complex HBsAg which is continuously produced from episomal cccDNA even when HBV replication subsides.
- S and L HBV proteins on the cell surface makes them accessible for antibodies which are the hallmark of seroconversion when patients recover from the acute phase of infections and change from circulating HBsAg to antiHBs. If seroconversion does not occur, up to 30 % of hepatocytes continue to express HBV S protein also after highly active antiviral therapy of long duration. Thus beyond T lymphocytes recognizing specifically intracellular ⁇ processed HBV peptides and presented by MHC molecules at the cell surface other forms of T cell engagement are feasible aimed at intact surface protein such as S and L antigens accessible in the outer cell membrane.
- T-cell receptors Using single chain antibody fragments recognizing hepatitis B virus small (S) and large (L) envelope proteins, artificial T-cell receptors have been generated which allow directing grafted T-cells to infected hepatocytes and upon antigen contact activation of these T-cells to secrete cytokines and kill infected hepatocytes.
- S hepatitis B virus small
- L large envelope proteins
- bispecific single chain antibody molecules comprising a first domain with a binding specificity for the human and the non-chimpanzee primate CD3 epsilon antigen (as provided herein in context of this invention) as well as a second domain with a binding specificity for HBV or HCV envelope proteins of infected hepatocytes may be generated and are within the scope of this invention.
- the second binding domain binds to a cell surface antigen of a human and/or a non-chimpanzee primate.
- a cell surface antigen of a human and/or a non-chimpanzee primate e.g. bispecific single chain antibodies as defined herein
- monoclonal antibodies binding to both of the respective human and/or non-chimpanzee primate cell surface antigens can be utilized.
- Appropriate binding domains for the bispecific polypeptide as defined herein e.g. can be derived from cross-species specific monoclonal antibodies by recombinant methods described in the art.
- a monoclonal antibody binding to a human cell surface antigen and to the homolog of said cell surface antigen in a non-chimpanzee primate can be tested by FACS assays as set forth above. It is evident to those skilled in the art that cross-species specific antibodies can also be generated by hybridoma techniques described in the literature (Milstein and K ⁇ hler, Nature 256 (1975), 495-7). For example, mice may be alternately immunized with human and non-chimpanzee primate CD33. From these mice, cross- species specific antibody-producing hybridoma cells are isolated via hybridoma technology and analysed by FACS as set forth above. The generation and analysis of bispecific polypeptides such as bispecific single chain antibodies exhibiting cross- species specificity as described herein is shown in the following examples. The advantages of the bispecific single chain antibodies exhibiting cross-species specificity include the points enumerated below.
- the first binding domain capable of binding to an epitope of human and non-chimpanzee primate CD3 ⁇ chain comprises a VL region comprising CDR-L1 , CDR-L2 and CDR-L3 selected from:
- variable regions i.e. the variable light chain (“L” or “VL”) and the variable heavy chain (“H” or “VH”) are understood in the art to provide the binding domain of an antibody.
- This variable regions harbor the complementary determining regions.
- complementary determining region CDR
- CDR-L or L CDR refers to CDRs in the VL
- CDR-H or H CDR refers to the CDRs in the VH.
- the first binding domain capable of binding to an epitope of human and non-chimpanzee primate CD3 ⁇ chain comprises a VH region comprising CDR-H 1 , CDR-H2 and CDR- H 3 selected from: (a) CDR-H1 as depicted in SEQ ID NO. 12, CDR-H2 as depicted in SEQ ID NO.
- the binding domain capable of binding to an epitope of human and non-chimpanzee primate CD3 ⁇ chain comprises a VL region selected from the group consisting of a VL region as depicted in SEQ ID NO. 35, 39, 125, 129, 161 or 165.
- the first binding domain capable of binding to an epitope of human and non-chimpanzee primate CD3 ⁇ chain comprises a VH region selected from the group consisting of a VH region as depicted in SEQ ID NO. 15, 19, 33, 37, 51 , 55, 69, 73, 87, 91 , 105, 109, 123, 127, 141 , 145, 159, 163, 177 or 181.
- the polypeptide of the invention is characterized by the first binding domain capable of binding to an epitope of human and non-chimpanzee primate CD3 ⁇ chain, which comprises a VL region and a VH region selected from the group consisting of: (a) a VL region as depicted in SEQ ID NO. 17 or 21 and a VH region as depicted in SEQ ID NO. 15 or 19; (b) a VL region as depicted in SEQ ID NO. 35 or 39 and a VH region as depicted in SEQ ID NO. 33 or 37;
- T a VL region as depicted in SEQ ID NO. 107 or 111 and a VH region as depicted in SEQ ID NO. 105 or 109;
- G a VL region as depicted in SEQ ID NO. 125 or 129 and a VH region as depicted in SEQ ID NO. 123 or 127;
- H a VL region as depicted in SEQ ID NO. 143 or 147 and a VH region as depicted in SEQ ID NO. 141 or 145;
- the pairs of VH-regions and VL-regions are in the format of a single chain antibody (scFv).
- the VH and VL regions are arranged in the order VH-VL or VL-VH. It is preferred that the VH-region is positioned N-terminally to a linker sequence.
- the VL-region is positioned C-terminally of the linker sequence.
- a preferred embodiment of the above described polypeptide of the invention is characterized by the first binding domain capable of binding to an epitope of human and non-chimpanzee primate CD3 ⁇ chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 25, 41 , 43, 59, 61 , 77, 79, 95, 97, 113, 115, 131 , 133, 149, 151 , 167, 169, 185 or 187.
- the invention further relates to an above described polypeptide, wherein the second binding domain binds to a cell surface antigen, which is preferably a tumor antigen.
- tumor antigen as used herein may be understood as those antigens that are presented on tumor cells. These antigens can be presented on the cell surface with an extracellular part, which is often combined with a transmembrane and cytoplasmic part of the molecule. These antigens can sometimes be presented only by tumor cells and never by the normal ones. Tumor antigens can be exclusively expressed on tumor cells or might represent a tumor specific mutation compared to normal cells. In this case, they are called tumor-specific antigens. More common are antigens that are presented by tumor cells and normal cells, and they are called tumor-associated antigens.
- tumor-associated antigens can be overexpressed compared to normal cells or are accessible for antibody binding in tumor cells due to the less compact structure of the tumor tissue compared to normal tissue.
- tumor antigens as used herein are EGFR (Liu, Br. J. Cancer 82/12 (2000), 1991-1999; Bonner, Semin. Radiat. Oncol. 12 (2002), 11-20; Kiyota, Oncology 63/1 (2002), 92-98; Kuan, Brain Tumor Pathol. 17/2 (2000), 71-78), EGFRvIII (Kuan, Brain Tumor Pathol. 17/2 (2000), 71-78), Carboanhydrase IX (MN/CA IX) (Uemura, Br. J.
- the tumor antigen is selected from EGFR, EGFRvIII, MCSP, EpCAM, Carbonic anhydrase IX (CAIX), CD30, CD33, Her2/neu, CD44v6 and Muc- 1.
- EGFR also known as c-erb1 or HER1 belongs to the erbB receptor tyrosine kinase family. When activated by binding of a ligand from the EGF family of growth factors, EGFR homodimerizes or heterodimerizes with a second EGFR or another member of the erbB receptor family, respectively, initiating a signaling cascade through mitogen- activated protein kinases and other transcription factors leading to proliferation, differentiation and repair (Olayioye, EMBO J. 19 (2000), 3159-67). EGFR is overexpressed in many epithelial cancers, including colorectal, breast, lung, and head and neck cancers (Mendelsohn, J. Clin. Oncol.
- Monoclonal antibodies that target the extracellular ligand binding domain or the intracellular tyrosine kinase signaling cascade of EGFR have been shown efficacy as antitumor target (Laskin, Cancer Treat. Review 30 (2004), 1-17).
- cetuximab a humanized monoclonal antibody to EGFR, which competitively inhibits the extracellular domain of EGFR to inhibit ligand activation of the receptor, was approved by the Food and Drug Administration (FDA) in 2004 for the treatment of metastatic colon cancer in combination with the topoisomerase inhibitor irinotecan.
- MCSP Melanoma-associated cell surface chondroitin sulphate proteoglycane
- HMW-MAA high-molecular weight melanoma-associated antigen
- melanoma-associated proteoglycan is a large cell surface proteoglycan of more than 450 kDa comprising a 250 kDa glycoprotein core and glycosaminoglycan chains attached to it (Pluschke, PNAS 93 (1996), 9710; Nishiyama, J. Cell Biol. 114 (1991), 359).
- HMW-MAA high-molecular weight melanoma-associated antigen
- melanoma-associated proteoglycan is a large cell surface proteoglycan of more than 450 kDa comprising a 250 kDa glycoprotein core and glycosaminoglycan chains attached to it (Pluschke, PNAS 93 (1996), 9710; Nishiyama, J. Cell Biol. 114 (1991), 359)
- MCSP extracellular matrix
- MCSP activation can enhance integrin- mediated cell spreading by cdc42- and p130cas-dependent mechanisms.
- MCSP associates through chondroitin sulphate with MT3-MMP, which can degrade various ECM-proteins. It may specifically localize MT3-MMP to cellular ECM-adhesion sites. Thus, both molecules seem to be required for invasion of type I collagen and degradation of type I gelatine (lida (2001), loc. cit.).
- MSCP is a major marker for melanoma, where it is highly expressed on the surface of the tumor cells. It is expressed by at least 90% of melanoma lesions (Natali, J. Natl. Cancer Inst. 72 (1984), 13). Among all melanoma associated antigens, MCSP displays the lowest degree of heterogeneity (Natali, Cancer Res. 45 (1985), 2883).
- polypeptide is a bispecific single chain antibody molecule.
- the drug candidate is a bispecific antibody, e.g. a bispecific single chain antibody.
- a bispecific antibody requires that both antigens recognized are cross-species specific with a given animal species to allow for safety testing in such animal.
- the present invention provides polypeptides comprising a first binding domain capable of binding to an epitope of human and non- chimpanzee primate CD3 ⁇ chain and a second binding domain capable of binding to a cell surface antigen, wherein the second binding domain preferably also binds to a cell surface antigen of a human and a non-chimpanzee primate.
- the advantage of bispecific single chain antibody molecules as drug candidates fulfilling the requirements of the preferred polypeptide of the invention is the use of such molecules in preclinical animal testing as well as in clinical studies and even for therapy in human.
- the second binding domain binding to a cell surface antigen is of human origin.
- cross-species specific bispecific molecule In a cross-species specific bispecific molecule according to the invention the binding domain binding to an epitope of human and non-chimpanzee primate CD3 epsilon chain is located in the order VH-VL or VL-VH at the N-terminus or the C-terminus of the bispecific molecule.
- Examples for cross- species specific bispecific molecules according to the invention in different arrangements of the VH- and the VL-chain in the first and the second binding domain are described in the appended examples.
- a "bispecific single chain antibody” denotes a single polypeptide chain comprising two binding domains.
- Each binding domain comprises one variable region from an antibody heavy chain ("VH region"), wherein the VH region of the first binding domain specifically binds to the CD3 ⁇ molecule, and the VH region of the second binding domain specifically binds to a cell surface antigen, as defined in more detail below.
- the two binding domains are optionally linked to one another by a short polypeptide spacer.
- a non-limiting example for a polypeptide spacer is GIy-GIy-GIy- Gly-Ser (G-G-G-G-S) and repeats thereof.
- Each binding domain may additionally comprise one variable region from an antibody light chain ("VL region"), the VH region and VL region within each of the first and second binding domains being linked to one another via a polypeptide linker, for example of the type disclosed and claimed in EP 623679 B1 , but in any case long enough to allow the VH region and VL region of the first binding domain and the VH region and VL region of the second binding domain to pair with one another such that, together, they are able to specifically bind to the respective first and second molecules.
- VL region antibody light chain
- an above characterized bispecific single chain antibody molecule comprises a group of the following sequences as CDR H1 , CDR H2, CDR H3, CDR L1 , CDR L2 and CDR L3 in the second binding domain selected from the group consisting of: SEQ ID NOs: 189-194, SEQ ID NOs: 201-206, SEQ ID NOs: 219-224, SEQ ID NOs: 237-242, SEQ ID NOs:255-260, SEQ ID NOs:273-279, SEQ ID NOs: 382-384 and 387-389, SEQ ID NOs: 400-402 and 405-407, SEQ ID NOs: 418-420 and 423-425, SEQ ID NOs: 436-438 and 441-443, SEQ ID NOs: 454-456 and 459-461 , SEQ ID NOs: 472-474 and 477-479, SEQ ID NOs: 490-492 and 495-497, SEQ ID NOs: and 508-510 and 51
- a particularly preferred embodiment of the invention concerns an above characterized polypeptide, wherein the bispecific single chain antibody molecule comprises a sequence selected from: (a) an amino acid sequence as depicted in any of SEQ ID NOs. 291 , 293, 295, 297, 299, 301 , 303, 305, 307, 309, 311 , 313, 315, 317, 319, 321 , 323, 325, 327, 329, 331 , 333, 335, 337, 339, 341 , 343, 345 or 347, 393, 395, 397, 411 , 413, 415, 429, 431 , 433, 447, 449, 451 , 465, 467, 469, 483, 485, 487, 501 , 503, 505, 519, 521 , 523, 1336, 1338, 1340, 538, 540, 542, 556, 570, 572, 574, 588, 590, 592, 626,
- the bispecific single chain antibodies are cross-species specific for CD3 epsilon and for the cell surface antigen recognized by their second binding domain.
- these bispecific single chain antibodies are cross-species specific for human and non-chimpanzee primate CD3 epsilon and for human and non-chimpanzee primate MCSP, EGFR, EGFRvIII, Carbonic anhydrase IX, CD30, CD33, IgE, CD44v6, Muc-1 , HBV and HCV.
- the present invention provides a nucleic acid sequence encoding an above described polypeptide of the invention.
- the present invention also relates to a vector comprising the nucleic acid molecule of the present invention.
- plasmids are known to those skilled in molecular biology, the choice of which would depend on the function desired and include plasmids, cosmids, viruses, bacteriophages and other vectors used conventionally in genetic engineering. Methods which are well known to those skilled in the art can be used to construct various plasmids and vectors; see, for example, the techniques described in Sambrook et al. (loc cit.) and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989), (1994). Alternatively, the polynucleotides and vectors of the invention can be reconstituted into liposomes for delivery to target cells.
- a cloning vector was used to isolate individual sequences of DNA. Relevant sequences can be transferred into expression vectors where expression of a particular polypeptide is required.
- Typical cloning vectors include pBluescript SK, pGEM, pUC9, pBR322 and pGBT9.
- Typical expression vectors include pTRE, pCAL-n-EK, pESP-1 , pOP13CAT.
- control sequence refers to DNA sequences, which are necessary to effect the expression of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism. In prokaryotes, control sequences generally include promoter, ribosomal binding site, and terminators. In eukaryotes generally control sequences include promoters, terminators and, in some instances, enhancers, transactivators or transcription factors.
- control sequence is intended to include, at a minimum, all components the presence of which are necessary for expression, and may also include additional advantageous components.
- operably linked refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
- a control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
- the control sequence is a promoter, it is obvious for a skilled person that double-stranded nucleic acid is preferably used.
- the recited vector is preferably an expression vector.
- An "expression vector” is a construct that can be used to transform a selected host and provides for expression of a coding sequence in the selected host. Expression vectors can for instance be cloning vectors, binary vectors or integrating vectors.
- Expression comprises transcription of the nucleic acid molecule preferably into a translatable mRNA.
- Regulatory elements ensuring expression in prokaryotes and/or eukaryotic cells are well known to those skilled in the art. In the case of eukaryotic cells they comprise normally promoters ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript.
- Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the P L , lac, trp or tac promoter in E.
- regulatory elements permitting expression in eukaryotic host cells are the A0X1 or G AU promoter in yeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells.
- Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide.
- transcription termination signals such as the SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide.
- leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium may be added to the coding sequence of the recited nucleic acid sequence and are well known in the art; see also the appended Examples.
- the leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a portion thereof, into the periplasmic space or extracellular medium.
- the heterologous sequence can encode a fusion protein including an. N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product; see supra.
- suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM ⁇ , pRc/CMV, pcDNAI , pcDNA3 (In-vitrogene), pEF-DHFR, pEF-ADA or pEF-neo (Mack et al. PNAS (1995) 92, 7021- 7025 and Haut et al.
- the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming of transfecting eukaryotic host cells, but control sequences for prokaryotic hosts may also be used.
- the vector Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and as desired, the collection and purification of the polypeptide of the invention may follow; see, e.g., the appended examples.
- An alternative expression system which can be used to express a cell cycle interacting protein is an insect system.
- Autographa califomica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in T ⁇ choplusia larvae.
- the coding sequence of a recited nucleic acid molecule may be cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of said coding sequence will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein coat. The recombinant viruses are then used to infect S.
- frugiperda cells or Thchoplusia larvae in which the protein of the invention is expressed (Smith, J. Virol. 46 (1983), 584; Engelhard, Proc. Nat. Acad. Sci. USA 91 (1994), 3224-3227).
- Additional regulatory elements may include transcriptional as well as translational enhancers.
- the above-described vectors of the invention comprise a selectable and/or scorable marker.
- Selectable marker genes useful for the selection of transformed cells and, e.g., plant tissue and plants are well known to those skilled in the art and comprise, for example, antimetabolite resistance as the basis of selection for dhfr, which confers resistance to methotrexate (Reiss, Plant Physiol. (Life Sci. Adv.) 13 (1994), 143-149); npt, which confers resistance to the aminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella, EMBO J. 2 (1983), 987-995) and hygro, which confers resistance to hygromycin (Marsh, Gene 32 (1984), 481-485).
- trpB which allows cells to utilize indole in place of tryptophan
- hisD which allows cells to utilize histinol in place of histidine
- mannose-6-phosphate isomerase which allows cells to utilize mannose
- ODC ornithine decarboxylase
- DFMO deaminase from Aspergillus terreus which confers resistance to Blasticidin S (Tamura, Biosci. Biotechnol. Biochem. 59 (1995), 2336-2338).
- luciferase Giacomin, Pl. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121), green fluorescent protein (Gerdes, FEBS Lett. 389 (1996), 44-47) or ⁇ -glucuronidase (Jefferson, EMBO J. 6 (1987), 3901-3907).
- This embodiment is particularly useful for simple and rapid screening of cells, tissues and organisms containing a recited vector.
- nucleic acid molecule can be used alone or as part of a vector to express the polypeptide of the invention in cells, for, e.g., purification but also for gene therapy purposes.
- the nucleic acid molecules or vectors containing the DNA sequence(s) encoding any one of the above described polypeptide of the invention is introduced into the cells which in turn produce the polypeptide of interest.
- Gene therapy which is based on introducing therapeutic genes into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer.
- Suitable vectors, methods or gene-delivery systems for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813; Verma, Nature 389 (1994), 239; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Onodera, Blood 91 (1998), 30-36; Verma, Gene Ther. 5 (1998), 692-699; Nabel, Ann. N.Y. Acad. Sci.
- nucleic acid molecules and vectors may be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g., adenoviral, retroviral) into the cell.
- said cell is a germ line cell, embryonic cell, or egg cell or derived there from, most preferably said cell is a stem cell.
- An example for an embryonic stem cell can be, inter alia, a stem cell as described in Nagy, Proc. Natl. Acad. Sci. USA 90 (1993), 8424-8428.
- the invention also provides for a host transformed or transfected with a vector of the invention.
- Said host may be produced by introducing the above described vector of the invention or the above described nucleic acid molecule of the invention into the host.
- the presence of at least one vector or at least one nucleic acid molecule in the host may mediate the expression of a gene encoding the above described single chain antibody constructs.
- the described nucleic acid molecule or vector of the invention, which is introduced in the host may either integrate into the genome of the host or it may be maintained extrachromosomally.
- the host can be any prokaryote or eukaryotic cell.
- prokaryote is meant to include all bacteria, which can be transformed or transfected with DNA or RNA molecules for the expression of a protein of the invention.
- Prokaryotic hosts may include gram negative as well as gram positive bacteria such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis.
- eukaryotic is meant to include yeast, higher plant, insect and preferably mammalian cells.
- the protein encoded by the polynucleotide of the present invention may be glycosylated or may be non-glycosylated.
- the length of said FLAG-tag is about 4 to 8 amino acids, most preferably 8 amino acids.
- said the host is a bacterium or an insect, fungal, plant or animal cell. It is particularly envisaged that the recited host may be a mammalian cell. Particularly preferred host cells comprise CHO cells, COS cells, myeloma cell lines like SP2/0 or NS/0. As illustrated in the appended examples, particularly preferred are CHO-cells as hosts.
- said host cell is a human cell or human cell line, e.g. per.c ⁇ (Kroos, Biotechnol. Prog., 2003, 19:163-168).
- the present invention thus relates to a process for the production of a polypeptide of the invention, said process comprising culturing a host of the invention under conditions allowing the expression of the polypeptide of the invention and recovering the produced polypeptide from the culture.
- the transformed hosts can be grown in fermentors and cultured according to techniques known in the art to achieve optimal cell growth.
- the polypeptide of the invention can then be isolated from the growth medium, cellular lysates, or cellular membrane fractions.
- the isolation and purification of the, e.g., microbially expressed polypeptides of tke i «we»tion may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies directed, e.g., against a tag of the polypeptide of the invention or as described in the appended examples.
- the polypeptide of the invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like; see, Scopes, "Protein Purification", Sphnger-Verlag, N.Y. (1982). Substantially pure polypeptides of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity are most preferred, for pharmaceutical uses. Once purified, partially or to homogeneity as desired, the polypeptide of the invention may then be used therapeutically (including extracorporeal ⁇ ) or in developing and performing assay procedures. Furthermore, examples for methods for the recovery of the polypeptide of the invention from a culture are described in detail in the appended examples.
- the invention provides for a composition comprising a polypeptide of the invention or a polypeptide as produced by the process disclosed above.
- said composition is a pharmaceutical composition.
- the term "pharmaceutical composition” relates to a composition for administration to a patient, preferably a human patient.
- the particular preferred pharmaceutical composition of this invention comprises binding molecules directed against and generated against context-independent CD3 epitopes.
- the pharmaceutical composition comprises suitable formulations of carriers, stabilizers and/or excipients.
- the pharmaceutical composition comprises a composition for parenteral, transdermal, intraluminal, intraarterial, intrathecal and/or intranasal administration or by direct injection into tissue. It is in particular envisaged that said composition is administered to a patient via infusion or injection.
- Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
- the present invention provides for an uninterrupted administration of the suitable composition.
- uninterrupted, i.e. continuous administration may be realized by a small pump system worn by the patient for metering the influx of therapeutic agent into the body of the patient.
- the pharmaceutical composition comprising the binding molecules directed against and generated against context-independent CD3 epitopes of the invention can be administered by using said pump systems.
- Such pump systems are generally known in the art, and commonly rely on periodic exchange of cartridges containing the therapeutic agent to be infused.
- the continuous or uninterrupted administration of these binding molecules directed against and generated against context-independent CD3 epitopes of this invention may be intravenuous or subcutaneous by way of a fluid delivery device or small pump system including a fluid driving mechanism for driving fluid out of a reservoir and an actuating mechanism for actuating the driving mechanism.
- Pump systems for subcutaneous administration may include a needle or a cannula for penetrating the skin of a patient and delivering the suitable composition into the patient's body.
- Said pump systems may be directly fixed or attached to the skin of the patient independently of a vein, artery or blood vessel, thereby allowing a direct contact between the pump system and the skin of the patient.
- the pump system can be attached to the skin of the patient for 24 hours up to several days.
- the pump system may be of small size with a reservoir for small volumes.
- the volume of the reservoir for the suitable pharmaceutical composition to be administered can be between 0.1 and 50 ml.
- the continuous administration may be transdermal by way of a patch worn on the skin and replaced at intervals.
- transdermal administration is especially amenable to uninterrupted administration, as exchange of a first exhausted patch can advantageously be accomplished simultaneously with the placement of a new, second patch, for example on the surface of the skin immediately adjacent to the first exhausted patch and immediately prior to removal of the first exhausted patch. Issues of flow interruption or power cell failure do not arise.
- composition of the present invention comprising in particular binding molecules directed against and generated against context-independent CD3 epitopes may further comprise a pharmaceutically acceptable carrier.
- suitable pharmaceutical carriers include solutions, e.g. phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions, liposomes, etc.
- Compositions comprising such carriers can be formulated by well known conventional methods.
- Formulations can comprise carbohydrates, buffer solutions, amino acids and/or surfactants.
- Carbohydrates may be non-reducing sugars, preferably trehalose, sucrose, octasulfate, sorbitol or xylitol.
- Such formulations may be used for continuous administrations which may be intravenuous or subcutaneous with and/or without pump systems.
- Amino acids may be charged amino acids, preferably lysine, lysine acetate, arginine, glutamate and/or histidine.
- Surfactants may be detergents, preferably with a molecular weight of >1.2 KD and/or a polyether, preferably with a molecular weight of >3 KD.
- Non-limiting examples for preferred detergents are Tween 20, Tween 40, Tween 60, Tween 80 or Tween 85.
- Non-limiting examples for preferred polyethers are PEG 3000, PEG 3350, PEG 4000 or PEG 5000.
- Buffer systems used in the present invention can have a preferred pH of 5-9 and may comprise citrate, succinate, phosphate, histidine and acetate.
- the compositions of the present invention can be administered to the subject at a suitable dose which can be determined e.g. by dose escalating studies by administration of increasing doses of the polypeptide of the invention exhibiting cross-species specificity described herein to non-chimpanzee primates, for instance macaques.
- the polypeptide of the invention exhibiting cross-species specificity described herein can be advantageously used in identical form in preclinical testing in non-chimpanzee primates and as drug in humans.
- These compositions can also be administered in combination with other proteinaceous and non-proteinaceous drugs.
- These drugs may be administered simultaneously with the composition comprising the polypeptide of the invention as defined herein or separately before or after administration of said polypeptide in timely defined intervals and doses.
- the dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
- nonaqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
- Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
- Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
- Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
- compositions of the present invention might comprise proteinaceous carriers, like, e.g., serum albumin or immunoglobulin, preferably of human origin. It is envisaged that the composition of the invention might comprise, in addition to the polypeptide of the invention defined herein, further biologically active agents, depending on the intended use of the composition. Such agents might be drugs acting on the gastro-intestinal system, drugs acting as cytostatica, drugs preventing hyperurikemia, drugs inhibiting immunoreactions (e.g.
- cytokines drugs modulating the inflammatory response
- drugs acting on the circulatory system drugs acting on the circulatory system and/or agents such as cytokines known in the art.
- the biological activity of the pharmaceutical composition defined herein can be determined for instance by cytotoxicity assays, as described in the following examples, in WO 99/54440 or by Schlereth et al. (Cancer Immunol. Immunother. 20 (2005), 1 - 12).
- "Efficacy” or "in vivo efficacy” as used herein refers to the response to therapy by the pharmaceutical composition of the invention, using e.g. standardized NCI response criteria.
- the success or in vivo efficacy of the therapy using a pharmaceutical composition of the invention refers to the effectiveness of the composition for its intended purpose, i.e.
- the in vivo efficacy may be monitored by established standard methods for the respective disease entities including, but not limited to white blood cell counts, differentials, Fluorescence Activated Cell Sorting, bone marrow aspiration.
- various disease specific clinical chemistry parameters and other established standard methods may be used.
- computer-aided tomography, X-ray, nuclear magnetic resonance tomography e.g.
- positron-emission tomography scanning white blood cell counts, differentials, Fluorescence Activated Cell Sorting, bone marrow aspiration, lymph node biopsies/histologies, and various lymphoma specific clinical chemistry parameters (e.g. lactate dehydrogenase) and other established standard methods may be used.
- a pharmacokinetic profile of the drug candidate i.e. a profile of the pharmacokinetic parameters that effect the ability of a particular drug to treat a given condition
- Pharmacokinetic parameters of the drug influencing the ability of a drug for treating a certain disease entity include, but are not limited to: half-life, volume of distribution, hepatic first-pass metabolism and the degree of blood serum binding.
- the efficacy of a given drug agent can be influenced by each of the parameters mentioned above.
- Hepatic first-pass metabolism is meant the propensity of a drug to be metabolized upon first contact with the liver, i.e. during its first pass through the liver.
- Volume of distribution means the degree of retention of a drug throughout the various compartments of the body, like e.g. intracellular and extracellular spaces, tissues and organs, etc. and the distribution of the drug within these compartments.
- Degree of blood serum binding means the propensity of a drug to interact with and bind to blood serum proteins, such as albumin, leading to a reduction or loss of biological activity of the drug.
- Pharmacokinetic parameters also include bioavailability, lag time (Tlag), Tmax, absorption rates, more onset and/or Cmax for a given amount of drug administered.
- Bioavailability means the amount of a drug in the blood compartment.
- Lag time means the time delay between the administration of the drug and its detection and measurability in blood or plasma.
- Tmax is the time after which maximal blood concentration of the drug is reached, and
- Cmax is the blood concentration maximally obtained with a given drug. The time to reach a blood or tissue concentration of the drug which is required for its biological effect is influenced by all parameters.
- Pharmacokinetik parameters of bispecific single chain antibodies, a preferred embodiment of the polypeptide of the invention, exhibiting cross-species specificity, which may be determined in preclinical animal testing in non-chimpanzee primates as outlined above are also set forth e.g. in the publication by Schlereth et al. (Cancer Immunol. Immunother. 20 (2005), 1 - 12).
- toxicity refers to the toxic effects of a drug manifested in adverse events or severe adverse events. These side events might refer to a lack of tolerability of the drug in general and/or a lack of local tolerance after administration. Toxicity could also include teratogenic or carcinogenic effects caused by the drug.
- safety means the administration of a drug without inducing severe adverse events directly after administration (local tolerance) and during a longer period of application of the drug.
- Safety can be evaluated e.g. at regular intervals during the treatment and follow-up period. Measurements include clinical evaluation, e.g. organ manifestations, and screening of laboratory abnormalities. Clinical evaluation may be carried out and deviating to normal findings recorded/coded according to NCI-CTC and/or MedDRA standards.
- Organ manifestations may include criteria such as allergy/immunology, blood/bone marrow, cardiac arrhythmia, coagulation and the like, as set forth e.g. in the Common Terminology Criteria for adverse events v3.0 (CTCAE).
- Laboratory parameters which may be tested include for instance haematology, clinical chemistry, coagulation profile and urine analysis and examination of other body fluids such as serum, plasma, lymphoid or spinal fluid, liquor and the like.
- Safety can thus be assessed e.g. by physical examination, imaging techniques (i.e. ultrasound, x-ray, CT scans, Magnetic Resonance Imaging (MRI), other measures with technical devices (i.e. electrocardiogram), vital signs, by measuring laboratory parameters and recording adverse events.
- adverse events in non-chimpanzee primates in the uses and methods according to the invention may be examined by histopathological and/or histochemical methods.
- effective and non-toxic dose refers to a tolerable dose of the bispecific single chain antibody as defined herein which is high enough to cause depletion of pathologic cells, tumor elimination, tumor shrinkage or stabilization of disease without or essentially without major toxic effects.
- effective and non-toxic doses may be determined e.g. by dose escalation studies described in the art and should be below the dose inducing severe adverse side events (dose limiting toxicity, DLT).
- the invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising a polypeptide of this invention (i.e. a polypeptide comprising at least one binding domain capable of binding to an epitope of human and non-chimpanzee primate CD3 epsilon chain, wherein the epitope is part of an amino acid sequence comprised in the group consisting of SEQ ID NOs. 2, 4, 6, or 8 in accordance with this invention or produced according to the process according to the invention) for the prevention, treatment or amelioration of a disease selected from a proliferative disease, a tumorous disease, or an immunological disorder.
- said pharmaceutical composition further comprises suitable formulations of carriers, stabilizers and/or excipients.
- a further aspect of the invention relates to a use of a polypeptide as defined herein above or produced according to a process defined herein above, for the preparation of a pharmaceutical composition for the prevention, treatment or amelioration of a disease.
- said disease is a proliferative disease, a tumorous disease, or an immunological disorder. It is further preferred that said tumorous disease is a malignant disease, preferably cancer.
- said pharmaceutical composition is suitable to be administered in combination with an additional drug, i.e. as part of a co-therapy.
- an active agent may be optionally included in the same pharmaceutical composition as the polypeptide of the invention, or may be included in a separate pharmaceutical composition.
- said separate pharmaceutical composition is suitable for administration prior to, simultaneously as or following administration of said pharmaceutical composition comprising the polypeptide of the invention.
- the additional drug or pharmaceutical composition may be a non-proteinaceous compound or a proteinaceous compound.
- the additional drug is a proteinaceous compound, it is advantageous that the proteinaceous compound be capable of providing an activation signal for immune effector cells.
- said proteinaceous compound or non-proteinaceous compound may be administered simultaneously or non-simultaneously with the polypeptide of the invention, a nucleic acid molecule as defined hereinabove, a vector as defined as defined hereinabove, or a host as defined as defined hereinabove.
- Another aspect of the invention relates to a method for the prevention, treatment or amelioration of a disease in a subject in the need thereof, said method comprising the step of administration of an effective amount of a pharmaceutical composition of the invention.
- said disease is a proliferative disease, a tumorous disease, or an immunological disorder.
- said tumorous disease is a malignant disease, preferably cancer.
- said pharmaceutical composition is suitable to be administered in combination with an additional drug, i.e. as part of a co-therapy.
- an active agent may be optionally included in the same pharmaceutical composition as the polypeptide of the invention, or may be included in a separate pharmaceutical composition.
- said separate pharmaceutical composition is suitable for administration prior to, simultaneously as or following administration of said pharmaceutical composition comprising the polypeptide of the invention.
- the additional drug or pharmaceutical composition may be a non-proteinaceous compound or a proteinaceous compound.
- the additional drug is a proteinaceous compound, it is advantageous that the proteinaceous compound be capable of providing an activation signal for immune effector cells.
- said proteinaceous compound or non-proteinaceous compound may be administered simultaneously or non-simultaneously with the polypeptide of the invention, a nucleic acid molecule as defined hereinabove, a vector as defined as defined hereinabove, or a host as defined as defined hereinabove.
- said subject is a human.
- the invention relates to a kit comprising a polypeptide of the invention, a nucleic acid molecule of the invention, a vector of the invention, or a host of the invention.
- Item 1 A method for the identification of (a) polypeptide(s) comprising a cross- species specific binding domain capable of binding to an epitope of human and non- chimpanzee primate CD3 epsilon (CD3 ⁇ ), the method comprising the steps of: (a) contacting the polypeptide(s) with an N-terminal fragment of the extracellular domain of CD3 ⁇ of maximal 27 amino acids comprising the amino acid sequence Gln-Asp-Gly-Asn-Glu-Glu-Met-Gly (SEQ ID NO. 381) or Gln-Asp-Gly-Asn-Glu-Glu- Ne-GIy (SEQ ID NO. 382), fixed via its C-terminus to a solid phase; (b) eluting the bound polypeptide(s) from said fragment; and (c) isolating the polypeptide(s) from the eluate of (b).
- polypeptide(s) identified by the above method of the invention are of human origin.
- the present "method for the identification of (a) polypeptide(s)" is understood as a method for the isolation of one or more different polypeptides with the same specificity for the fragment of the extracellular domain of CD3 ⁇ comprising at its N- terminus the amino acid sequence Gln-Asp-Gly-Asn-Glu-Glu-Met-Gly (SEQ ID NO. 381) or Gln-Asp-Gly-Asn-Glu-Glu-lle-Gly (SEQ ID NO. 382) from a plurality of polypeptide candidates as well as a method for the purification of a polypeptide from a solution.
- Non-limiting embodiments of a method of the isolation of one or more different polypeptides with the same specificity for the fragment of the extracellular domain of CD3 ⁇ comprise methods for the selection of antigen-specific binding entities, e.g. panning methods as commonly used for hybridoma screening, screening of transiently/stably transfected clones of eukaryotic host cells or in phage display methods.
- a non-limiting example for the latter method for the purification of a polypeptide from a solution is e.g. the purification of a recombinantly expressed polypeptide from a culture supernatant or a preparation from such culture.
- the fragment used in the method of the invention is a N-terminal fragment of the extracellular domain of the primate CD3 ⁇ molecule.
- the amino acid sequence of the extracellular domain of the CD3 ⁇ molecule of different primates is depicted in SEQ ID NOs: 1 , 3, 5 and 7.
- the two forms of the N-terminal octamer are depicted in SEQ ID NOs: 381 and 382. It is prefered that this N-terminus is freely available for binding of the polypeptides to be identified by the method of the invention.
- the term "freely available" is understood in the context of the invention as free of additional motives such as a His-tag.
- the selection of the means and methods for the fixation/immobilization of the fragment to said solid support depend on the election of the solid support.
- a commonly used method for the fixation/immobilization is a coupling via an N- hydroxysuccinimide (NHS) ester.
- NHS N- hydroxysuccinimide
- the chemistry underlying this coupling as well as alternative methods for the fixation/immobilization are known to the person skilled in the art, e.g. from Hermanson "Bioconjugate Techniques", Academic Press, Inc. (1996).
- the fixation to/immobilization on chromatographic supports the following means are commonly used: NHS-activated sepharose (e.g. HiTrap-NHS of GE Life Science - Amersham), CnBr-activated sepharose (e.g.
- GE Life Science - Amersham NHS-activated dextran beads (Sigma) or activated polymethacrylate. These reagents may also be used in a batch approach. Moreover, dextran beads comprising iron oxide (e.g. available from Miltenyi) may be used in a batch approach. These beads may be used in combination with a magnet for the separation of the beads from a solution.
- Polypeptides can be immobilized on a Biacore chip (e.g. CM5 chips) by the use of NHS activated carboxymethyldextran. Further examples for an appropriate solid support are amine reactive MultiWell plates (e.g. Nunc ImmobilizerTM plates).
- said fragment of the extracellular domain of CD3 epsilon can be directly coupled to the solid support or via a stretch of amino acids, which might be a linker or another protein/polypeptide moiety.
- the extracellular domain of CD3 epsilon can be indirectly coupled via one or more adaptor molecule(s).
- a method for the isolation of one or more different polypeptides with the same specificity for the fragment of the extracellular domain of CD3 ⁇ comprising at its N-terminus the amino acid sequence Gln-Asp-Gly-Asn-Glu- GIu-X-GIy from a plurality of polypeptide candidates may comprise one or more steps of the following methods for the selection of antigen-specific entities: CD3 ⁇ specific binding molecules can be selected from antibody derived repertoires.
- a phage display library can be constructed based on standard procedures, as for example disclosed in "Phage Display: A Laboratory Manual”; Ed. Barbas, Burton, Scott & Silverman; Cold Spring Harbor Laboratory Press, 2001.
- the format of the antibody fragments in the antibody library can be scFv, but may generally also be a Fab fragment or even a single domain antibody fragment.
- na ⁇ ve antibody fragment libraries may be used.
- human antibody fragment libraries may be favourable for the direct selection of human antibody fragments. In some cases they may form the basis for synthetic antibody libraries (Knappik et al. J MoI. Biol. 2000, 296:57 ff).
- the corresponding format may be Fab, scFv (as described below) or domain antibodies (dAbs, as reviewed in Holt et al., Trends Biotechnol. 2003, 21 :484 ff).
- a corresponding format of the antibody library may be Fab, scFv (as described below) or single domain antibodies (VHH).
- VHH single domain antibodies
- Immunized animals may form the basis for the construction of immune antibody libraries.
- Such libraries comprise phage display libraries.
- Such libraries may be generally constructed based on standard procedures, as for example disclosed in "Phage Display: A Laboratory Manual”; Ed. Barbas, Burton, Scott & Silverman; Cold Spring Harbor Laboratory Press, 2001.
- the non-human antibodies can also be humanized via phage display due to the generation of more variable antibody libraries that can be subsequently enriched for binders during selection.
- a phage display approach any one of the pools of phages that displays the antibody libraries forms a basis to select binding entities using the respective antigen as target molecule.
- the central step in which antigen specific, antigen bound phages are isolated is designated as panning. Due to the display of the antibody fragments on the surface of the phages, this general method is called phage display.
- One preferred method of selection is the use of small proteins such as the filamentous phage N2 domain translationally fused to the N-terminus of the scFv displayed by the phage.
- Another display method known in the art, which may be used to isolate binding entities is the ribosome display method (reviewed in Groves & Osbourn, Expert Opin Biol Ther. 2005, 5:125 ff; Lipovsek & Pluckthun, J Immunol Methods 2004, 290:52 ff).
- a phage library carrying the cloned scFv-repertoire can be harvested from the respective culture supernatant by PEG (polyethyleneglycole).
- ScFv phage particles may be incubated with immobilized CD3 ⁇ Fc fusion protein.
- the immobilized CD3 ⁇ Fc fusion protein may be coated to a solid phase. Binding entities can be eluted and the eluate can be used for infection of fresh uninfected bacterial hosts.
- Bacterial hosts successfully transduced with a phagemid copy, encoding a human scFv- fragment, can be selected again for carbenicillin resistance and subsequently infected with e.g. VCMS 13 helper phage to start the second round of antibody display and in vitro selection. A total of 4 to 5 rounds of selections is carried out, normally.
- the binding of isolated binding entities can be tested on CD3epsilon positive Jurkat cells, HPBaII cells, PBMCs or transfected eukaryotic cells that carry the N-terminal CD3 ⁇ sequence fused to surface displayed EpCAM using a flow cytometric assay (see appended Example 4).
- Item 2 The method of item 1 , wherein the polypeptide(s) comprise(s) the identified binding domain as a first binding domain and a second binding domain capable of binding to a cell surface antigen.
- bispecific single chain antibodies For the generation of the second binding domain of the polypeptide identified by the method of the invention, e.g. bispecific single chain antibodies as defined herein, monoclonal antibodies binding to both of the respective human and non-chimpanzee primate cell surface antigens can be used.
- Appropriate binding domains for the bispecific polypeptide as defined herein e.g. can be derived from cross-species specific monoclonal antibodies by recombinant methods described in the art.
- a monoclonal antibody binding to a human cell surface antigen and to the homolog of said cell surface antigen in a non-chimpanzee primate can be tested by FACS assays as set forth above.
- Hybridoma techniques as described in the literature can also be used for the generation of cross-species specific antibodies.
- mice may be alternately immunized with human and non-chimpanzee primate CD33. From these mice, cross- species specific antibody-producing hybridoma cells can be isolated via hybridoma technology and analysed by FACS as set forth above.
- FACS FACS as set forth above.
- the generation and analysis of bispecific polypeptides such as bispecific single chain antibodies exhibiting cross- species specificity as described herein is shown in the following Examples. The advantages of the bispecific single chain antibodies exhibiting cross-species specificity include the points enumerated below.
- Item 3 The method of item 2, wherein the second binding domain binds to a cell surface antigen of a human and a non-chimpanzee primate.
- Item 4 The method of any of items f to 3, wherein the first binding domain is an antibody.
- Item 5 The method of item 4, wherein the antibody is a single chain antibody.
- Item 6 The method of any of items 2 to 5, wherein the second binding domain is an antibody.
- Item 7 The method of any of items 1 to 6, wherein the fragment of the extracellular domain of CD3 ⁇ consists of one or more fragments of a polypeptide having an amino acid sequence of any one depicted in SEQ ID NOs.2, 4, 6 or 8.
- Item 8 The method of item 7, wherein said fragment is 8, 9, 10, 11 , 12, 13, 14,
- Item 9 The method of any of items 1 to 8, wherein the method of identification is a method of screening a plurality of polypeptides comprising a cross-species specific binding domain binding to an epitope of human and non-chimpanzee primate CD3 ⁇ .
- Item 10 The method of any of items 1 to 8, wherein the method of identification is a method of purification/isolation of a polypeptide comprising a cross-species specific binding domain binding to an epitope of human and non-chimpanzee primate CD3 ⁇ .
- the generated cross-species specific binding domain is of human origin.
- Item 13 Use according to item 12, wherein the antibody is a single chain antibody.
- Item 14 Use according to items 12 to 13, wherein the antibody is a bispecific antibody.
- the antibody is a bispecific antibody.
- Recombinant techniques and methods in immunology are described e.g. in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 3 rd edition 2001 ; Lefkovits; Immunology Methods Manual; The Comprehensive Sourcebook of Techniques; Academic Press, 1997; Golemis; Protein-Protein Interactions: A Molecular Cloning Manual; Cold Spring Laboratory Press, 2002. Further literature concerning any one of the antibodies, methods, uses and compounds to be employed in accordance with the present invention may be retrieved from public libraries and databases, using for example electronic devices.
- the public database "Medline”, available on the Internet, may be utilized, for example under http://www.ncbi.nlm.nih.gov/PubMed/medline.html.
- Further databases and addresses such as http://www.ncbi.nlm.nih.gov/ or listed at the EMBL-services homepage under http://www.embl.de/services/index.html are known to the person skilled in the art and can also be obtained using, e. g., http://www. google.com.
- Figure 1 Fusion of the N-terminal amino acids 1-27 of primate CD3 epsilon to a heterologous soluble protein.
- the figure shows the average absorption values of quadruplicate samples measured in an ELISA assay detecting the presence of a construct consisting of the N-terminal amino acids 1-27 of the mature human CD3 epsilon chain fused to the hinge and Fc gamma portion of human IgGI and a C-terminal 6 Histidine tag in a supernatant of transiently transfected 293 cells.
- the first column labeled "27 aa huCD3E" shows the average absorption value for the construct
- the second column labeled "irrel. SN" shows the average value for a supernatant of 293 cells transfected with an irrelevant construct as negative control.
- the comparison of the values obtained for the construct with the values obtained for the negative control clearly demonstrates the presence of the recombinant construct.
- Figure 3 shows the average absorption values of quadruplicate samples measured in an ELISA assay detecting the presence of a construct consisting of the N-terminal amino acids 1-27 of the
- the figure shows the average absorption values of quadruplicate samples measured in an ELISA assay detecting the binding of the cross species specific anti-CD3 binding molecules in form of crude preparations of periplasmatically expressed single-chain antibodies to a construct comprising the N-terminal 1-27 amino acids of the mature human CD3 epsilon chain fused to the hinge and Fc gamma portion of human IgGI and a C-terminal His6 tag.
- the columns show from left to right the average absorption values for the specificities designated as A2J HLP, I2C HLP E2M HLP, F7O HLP, G4H HLP, H2C HLP 1 E1 L HLP, F12Q HLP, F6A HLP and H1 E HLP.
- the rightmost column labelled "neg. contr.” shows the average absorption value for the single-chain preparation of a murine anti-human CD3 antibody as negative control.
- the comparison of the values obtained for the anti-CD3 specificities with the values obtained for the negative control clearly demonstrates the strong binding of the anti-CD3 specificities to the N-terminal 1-27 amino acids of the mature human CD3 epsilon chain.
- the histogram overlays from left to right and top to bottom show the results for the transfectants expressing the constructs comprising the human 27 mer, marmoset 27 mer, tamarin 27 mer, squirrel monkey 27 mer and swine 27 mer respectively.
- the thin line represents a sample incubated with PBS with 2% FCS instead of anti-Flag M2 antibody as negative control and the bold line shows a sample incubated with the anti-Flag M2 antibody.
- the overlay of the histograms shows binding of the anti-Flag M2 antibody to the transfectants, which clearly demonstrates the expression of the recombinant constructs on the transfectants.
- Figure 6 Histogram overlays of different transfectants tested in a FACS assay detecting the binding of the cross-species specific anti-CD3 binding molecules in form of crude preparations of periplasmatically expressed single-chain antibodies to the N-terminal amino acids 1-27 of the human, marmoset, tamarin and squirrel monkey CD3 epsilon chain respectively fused to cynomolgus EpCAM.
- Figure 6A shows that
- the histogram overlays from left to right and top to bottom show the results for the transfectants expressing the 1-27 CD3-EpCAM comprising the swine 27 mer tested with the CD3 specific binding molecules designated H2C HLP, F12Q HLP, E2M HLP and G4H HLP respectively.
- the thin line represents a sample incubated with a single- chain preparation of a murine anti-human CD3-antibody as negative control and the bold line shows a sample incubated with the respective anti-CD3 binding molecules indicated.
- the overlays of the histograms show specific and strong binding of the tested anti-CD3 specificities of the fully cross- species specific human bispecific single chain antibodies to cells expressing the recombinant transmembrane fusion proteins comprising the N-terminal amino acids 1-27 of the human, marmoset, tamarin and squirrel monkey CD3 epsilon chain respectively fused to cynomolgus EpCAM and show therefore multi primate cross- species specificity of the anti-CD3 binding molecules.
- FACS assay for detection of human CD3 epsilon on transfected murine EL4 T cells Graphical analysis shows an overlay of histograms. The bold line shows transfected cells incubated with the anti-human CD3 antibody UCHT-1. The thin line represents cells incubated with a mouse IgGI isotype control. Binding of the anti CD3 antibody UCHT1 clearly shows expression of the human CD3 epsilon chain on the cell surface of transfected murine EL4 T cells.
- binding values are calculated using the following formula:
- sample means the value in arbitrary units of binding depicting the degree of binding of a specific anti-CD3 antibody to a specific alanine-mutant as shown in the Figure
- Sample means the geometric mean fluorescence value obtained for a specific anti-CD3 antibody assayed on a specific alanine-scanning transfectant, negjContr.
- the figure shows the results for cross-species specific anti CD3 antibody A2J HLP expressed as chimeric IgG molecule. Reduced binding activity is observed for mutations to alanine at position 4 (asparagine), at position 23 (threonine) and at position 25 (isoleucine). Complete loss of binding is observed for mutations to alanine at position 1 (glutamine), at position 2 (aspartate), at position 3 (glycine) and at position 5 (glutamate).
- Figure 8B The figure shows the results for cross-species specific anti CD3 antibody E2M HLP, expressed as chimeric IgG molecule.
- FIG. 8D shows the results for cross-species specific anti CD3 antibody F12Q HLP, tested as periplasmatically expressed single-chain antibody. Complete loss of binding is observed for mutations to alanine at position 1 (glutamine), at position 2 (aspartate), at position 3 (glycine) and at position 5 (glutamate).
- FACS assay detecting the binding of the cross-species specific anti-CD3 binding molecule H2C HLP to human CD3 with and without N-terminal His6 tag.
- Histogram overlays are performed of the EL4 cell line transfected with wild-type human CD3 epsilon chain (left histogram) or the human CD3 epsilon chain with N- terminal His6 tag (right histogram) tested in a FACS assay detecting the binding of cross-species specific binding molecule H2C HLP.
- Samples are incubated with an appropriate isotype control as negative control (thin line), anti-human CD3 antibody UCHT-1 as positive control (dotted line) and cross-species specific anti-CD3 antibody H2C HLP in form of a chimeric IgG molecule (bold line).
- Histogram overlays show comparable binding of the UCHT-1 antibody to both transfectants as compared to the isotype control demonstrating expression of both recombinant constructs. Histogram overlays also show binding of the anti-CD3 binding molecule H2C HLP only to the wild-type human CD3 epsilon chain but not to the His6-human CD3 epsilon chain. These results demonstrate that a free N- terminus is essential for binding of the cross-species specific anti-CD3 binding molecule H2C HLP.
- Figure 10 Saturation binding of EGFR-21-63 LH x H2C on human CD3 positive PBMC to determine the KD value of CD3 binding on cells by FACS analysis. The assay is performed as described in Example 7.
- FIG. 11 FACS binding analysis of designated cross-species specific bispecific single chain constructs to CHO cells transfected with human EGFR, human CD3+ T cell line HPB- ALL, CHO cells transfected with cynomolgus EGFR and a macaque T cell line 4119 LnPx.
- the FACS staining is performed as described in Example 12.
- the thick line represents cells incubated with 2 ⁇ g/ml purified protein that are subsequently incubated with the anti-his antibody and the PE labeled detection antibody.
- the thin histogram line reflects the negative control: cells only incubated with the anti-his antibody and the detection antibody.
- Cytotoxic activity induced by designated cross-species specific single chain constructs redirected to indicated target cell lines A) Stimulated CD4-/CD56- human PBMCs are used as effector cells, CHO cells transfected with human EGFR as target cells. B) The macaque T cell line 4119 LnPx are used as effector cells, CHO cells transfected with cynomolgus EGFR as target cells. The assay is performed as described in Example 13.
- Figure 13 Cytotoxic activity induced by designated cross-species specific single chain constructs redirected to indicated target cell lines.
- the FACS staining is performed as described in Example 17.
- the thick line represents cells incubated with 2 ⁇ g/ml purified protein that are subsequently incubated with the anti-his antibody and the PE labeled detection antibody.
- the thin histogram line reflects the negative control: cells only incubated with the anti-his antibody and the detection antibody.
- FIG. 15 FACS binding analysis of designated cross-species specific bispecific single chain constructs CHO cells transfected with the human MCSP D3, human CD3+ T cell line HPB-ALL, CHO cells transfected with cynomolgus MCSP D3 and a macaque T cell line 4119 LnPx.
- the FACS staining is performed as described in Example 17.
- the thick line represents cells incubated with 2 ⁇ g/ml purified protein that are subsequently incubated with the anti-his antibody and the PE labeled detection antibody.
- the thin histogram line reflects the negative control: cells only incubated with the anti-his antibody and the detection antibody.
- FACS binding analysis of designated cross-species specific bispecific single chain constructs CHO cells transfected with the human MCSP D3, human CD3+ T cell line HPB-ALL, CHO cells transfected with cynomolgus MCSP D3 and a macaque T cell line 4119 LnPx.
- the FACS staining is performed as described in Example 17.
- the thick line represents cells incubated with 2 ⁇ g/ml purified monomeric protein that are subsequently incubated with the anti-his antibody and the PE labeled detection antibody.
- the thin histogram line reflects the negative control: cells only incubated with the anti-his antibody and the detection antibody.
- Cytotoxicity activity induced by designated cross-species specific MCSP specific single chain constructs redirected to indicated target cell lines A) Stimulated CD4- /CD56- human PBMCs are used as effector cells, CHO cells transfected with human MCSP D3 as target cells. B) The macaque T cell line 4119 LnPx are used as effector cells, CHO cells transfected with cynomolgus MCSP D3 as target cells. The assay is performed as described in Example 18.
- Figure 20 Cytotoxicity activity induced by designated cross-species specific MCSP specific single chain constructs redirected to indicated target cell lines.
- Cytotoxicity activity induced by designated cross-species specific MCSP specific single chain constructs redirected to indicated target cell lines A) Stimulated CD4- /CD56- human PBMCs are used as effector cells, CHO cells transfected with human MCSP D3 as target cells. B) The macaque T cell line 4119 LnPx are used as effector cells, CHO cells transfected with cynomolgus MCSP D3 as target cells. The assay is performed as described in Example 18.
- CHO cells transfected with human MCSP are used as target cell line and stimulated CD4-/CD56- human PBMCs are used as effector cells.
- the assay is performed as described in Example 19.
- FIG 25 Complex T cell redistribution pattern (open squares) in B-NHL patient #20 (Table 4) without circulating CD19-positive target B cells (filled triangles), during ramp initiation of the CD19xCD3 infusion i.e. even gradual increase of flow-rate from almost zero to 15 ⁇ g/m 2 /24h during the first 24 hours of treatment.
- Absolute cell counts are given in 1000 cells per microliter blood.
- the first data point shows baseline counts immediately prior to the start of infusion.
- the CD19xCD3 dose is given in parentheses beside the patient number. T cells reappearing in the circulating blood after the initial redistribution triggered by the first exposure to CD19xCD3 are partially induced to redisappear from circulating blood again by still increasing levels of CD19xCD3 during the ramp phase.
- T and B cell counts during treatment with CD19xCD3 of B-NHL patient #13 who had a significant number of circulating CD19-positive target B (lymphoma) cells (filled triangles). Absolute cell counts are given in 1000 cells per microliter blood. The first data point shows baseline counts immediately prior to the start of infusion. The CD19xCD3 dose is given in parentheses beside the patient number. T cells (open squares) disappear completely from the circulation upon start of CD19xCD3 infusion and do not reappear until the circulating CD19-positive B (lymphoma) cells (filled triangles) are depleted from the peripheral blood.
- FIG. 28 Model of T cell adhesion to endothelial cells induced by monovalent binding to context dependent CD3 epitopes.
- Monovalent interaction of a conventional CD3 binding molecule to its context dependent epitope on CD3 epsilon can lead to an allosteric change in the conformation of CD3 followed by the recruitment of Nck2 to the cytoplasmic domain of CD3 epsilon (Gil et al. (2002) Cell 109: 901).
- Nck2 is directly linked to integrins via PINCH and ILK (Legate et al.
- Specific lysis of CD33- positive target cells was determined in a standard 51 Chromium release assay at increasing concentrations of CD33-AF5 VH-VL x I2C VH-VL. Assay duration was 18 hours.
- the macaque T cell line 4119 LnPx was used as source of effector cells.
- CHO cells transfected with cynomolgus CD33 served as target cells. Effector- to target cell ratio (E:T-ratio) was 10:1.
- the concentration of CD33-AF5 VH-VL x I2C VH-VL required for half-maximal target cell lysis (EC50) was calculated from the dose response curve with a value of 2.7 ng/ml.
- Figure 30 (A) Dose- and time-dependent depletion of CD33-positive monocytes from the peripheral blood of cynomolgus monkeys through intravenous continuous infusion of CD33-AF5 VH-VL x I2C VH-VL as described in Example 21. The percentage relative to baseline (i.e. 100%) of absolute circulating CD33-positive monocyte counts after the duration of treatment as indicated above the columns is shown for each of two cynomolgus monkeys per dose level. The dose level (i.e. infusion flow-rate) is indicated below the columns. No depletion of circulating CD33-positive monocytes was observed in animals 1 and 2 treated for 7 days at a dose of 30 ⁇ g/m 2 /24h.
- Specific lysis of MCSP- positive target cells was determined in a standard 51 Chromium release assay at increasing concentrations of MCSP-G4 VH-VL x I2C VH-VL. Assay duration was 18 hours.
- the macaque T cell line 4119 LnPx was used as source of effector cells.
- CHO cells transfected with cynomolgus MCSP served as target cells. Effector- to target cell ratio (E:T-ratio) was 10:1.
- the concentration of MCSP-G4 VH-VL x I2C VH-VL required for half-maximal target cell lysis (EC50) was calculated from the dose response curve with a value of 1.9 ng/ml.
- FIG. 32 Absence of initial episodes of drop and subsequent recovery of absolute T cell counts (i.e. redistribution) in peripheral blood of cynomolgus monkeys during the starting phase of intravenous infusion with the CD3 binding molecule MCSP-G4 VH- VL x I2C VH-VL recognizing an essentially context independent CD3 epitope. Absolute cell counts are given in 1000 cells per microliter blood. The first data point shows baseline counts immediately prior to the start of infusion. The MCSP-G4 VH- VL x I2C VH-VL dose is given in parentheses beside the animal number.
- FACS staining is performed as described in Example 23.4.
- the bold lines represent cells incubated with 5 ⁇ g/ml purified bispecific single chain construct or cell culture supernatant of transfected cells expressing the cross-species specific bispecific antibody constructs.
- the filled histograms reflect the negative controls. Supernatant of untransfected CHO cells was used as negative control.
- the overlay of the histograms shows specific binding of the construct to human and macaque CD33 and human and macaque
- the diagrams show results of chromium release assays measuring cytotoxic activity induced by designated cross-species specific CD33 specific single chain constructs redirected to the indicated target cell lines. Effector cells were also used as indicated. The assays are performed as described in Example 23.5. The diagrams clearly demonstrate for each construct the potent recruitment of cytotoxic activity of human and macaque effector cells against human and macaque CD33 transfected CHO cells, respectively.
- the diagrams show results of chromium release assays measuring cytotoxic activity induced by designated cross-species specific CAIX specific single chain constructs redirected to the indicated target cell lines. Effector cells were also used as indicated. The assays are performed as described in Example 24.6. The diagrams clearly demonstrate for each construct the potent recruitment of cytotoxic activity of human and macaque effector cells against human and macaque CAIX transfected CHO cells respectively.
- FIG. 37 Cytotoxic activity induced by designated EpCAM specific single chain constructs against human EpCAM transfected CHO cells using human T cells as effector cells. The assay was performed as described in the example section. The diagrams clearly demonstrate potent recruitment of cytotoxic activity by each construct. Effector cells: stimulated CD4/CD56 depleted human PBMC. Target cells: CHO transfected with human EpCAM.
- Figure 38 FACS binding analysis of designated bispecific single chain constructs to CHO cells transfected with human EpCAM (2a), untransfected CHO cells as EpCAM and CD3 negative cell line (2b), human PBMCs (2c) and the macaque T cell line 4119 LnPx (2d) respectively. Cells were incubated with supernatant containing the respective bispecific single chain construct. The FACS staining is performed as described in the example section. For each cross-species specific bispecific single chain construct the overlay of the histograms shows specific binding of the construct to human EpCAM and human and macaque CD3.
- a CHO-EpCAM (human)
- b CHO (untransfected)
- c human PBMCs
- d macaque 4119LnPx (CD3+).
- the FACS staining is performed as described in Example 26.3.
- the thick line represents cells incubated with 2 ⁇ g/ml purified monomeric protein that are subsequently incubated with the anti-his antibody and the PE labeled detection antibody.
- the thin histogram line reflects the negative control: cells only incubated with the anti-his antibody and the detection antibody.
- Cytotoxic activity induced by designated cross-species specific single chain constructs redirected to indicated target cell lines A) Stimulated CD4-/CD56- human PBMCs are used as effector cells, CHO cells transfected with human EGFRvIII as target cells. B) The macaque T cell line 4119 LnPx are used as effector cells, CHO cells transfected with cynomolgus EGFRvIII as target cells. The assay is performed as described in Example 26.3.
- FIG 41 FACS binding analysis of designated cross-species specific bispecific single chain constructs to J558L cells transfected with human IgE, human CD3+ T cell line HPB- ALL, J558L cells transfected with macaque IgE and a macaque T cell line 4119 LnPx.
- the FACS staining is performed as described in Example 27.2.
- the thick line represents cells incubated with cell culture supernatant of CHO cells transfected with designated cross-species specific bispecific single chain construct that are subsequently incubated with the anti-his antibody and the PE labeled detection antibody.
- the thin histogram line reflects the negative control: cells only incubated with the anti-his antibody and the detection antibody.
- Cytotoxic activity induced by designated cross-species specific single chain constructs redirected to indicated target cell lines A) Stimulated CD4-/CD56- human PBMCs are used as effector cells, J558L cells transfected with human IgE as target cells. B) The macaque T cell line 4119 LnPx are used as effector cells, J558L cells transfected with macaque IgE as target cells. The assay is performed as described in Example 27.2.
- HPB-ALL CHO cells transfected with macaque CD44 and macaque PBMC respectively.
- the FACS staining was performed as described in Example 28.6.
- the bold lines represent cells incubated with 5 ⁇ g/ml purified bispecific single chain construct.
- the filled histograms reflect the negative controls. PBS with 2 % FCS was used as negative control.
- the overlay of the histograms shows specific binding of the construct to human and macaque CD44 and human and macaque CD3.
- the diagram shows results of a chromium release assay measuring cytotoxic activity induced by designated cross-species specific CD44 specific single chain constructs redirected to CHO cells transfected with human CD44 as described in Example 28.1.
- the diagrams show results of chromium release assays measuring cytotoxic activity induced by designated cross-species specific CD30 specific single chain constructs redirected to the indicated target cell lines. Effector cells were also used as indicated. The assays were performed as described in Example 29.6. The diagrams clearly demonstrate for each construct the potent recruitment of cytotoxic activity of human and macaque effector cells against cells positive for human and macaque CD30, respectively.
- the diagrams show results of chromium release assays measuring cytotoxic activity induced by designated cross-species specific HER2 specific single chain constructs redirected to the indicated target cell lines. Effector cells were also used as indicated. The assays were performed as described in Example 30.6. The diagrams clearly demonstrate for each construct shown the potent recruitment of cytotoxic activity of human and macaque effector cells against human and macaque HER2 transfected CHO cells, respectively.
- the Western blot detecting the histidine ⁇ tag confirms the identity of the protein band in the eluate as the cross- species specific bispecific single chain molecule.
- the faint signal for the flow through sample in this sensitive detection method further shows the nearly complete capture of bispecific single chain molecules by the purification method.
- the Western blot detecting the histidine ⁇ tag confirms the identity of the protein band in the eluate as the cross- species specific bispecific single chain molecule.
- the faint signal for the flow through sample in this sensitive detection method further shows the nearly complete capture of bispecific single chain molecules by the purification method.
- Standard curve of AF5HLxl2CHL in 50% macaque monkey serum The upper diagram shows the standard curve generated for the assay as described in Example 32.2.
- the lower diagram shows results for quality control samples of AF5HLxl2CHL in 50% macaque monkey serum.
- the recovery rates are above 90% for the high and mid QC sample and above 80% for the low QC sample.
- the assay allows for detection of AF5HLxl2CHL in serum samples in the range from 10 ng/ml to 200 ng/ml (before dilution).
- the lower diagram shows results for quality control samples of MCSP-G4 HL x I2C HL in 50% macaque monkey serum.
- the recovery rates are above 98% for the high and mid QC sample and above 85% for the low QC sample.
- the assay allows for detection of MCSP-G4 HL x I2C HL in serum samples in the range from 10 ng/ml to 200 ng/ml (before dilution).
- Figure 55 FACS binding analysis of an anti-Flag antibody to CHO cells transfected with the 1- 27 N-terminal amino acids of CD3 epsilon of the designated species fused to cynomolgus EpCAM.
- the FACS staining was performed as described in Example 33.1.
- the bold lines represent cells incubated with the anti-Flag antibody.
- the filled histograms reflect the negative controls. PBS with 2 % FCS was used as negative control.
- the histograms show strong and comparable binding of the anti-Flag antibody to all transfectants indicating strong and equal expression of the transfected constructs.
- the centre histogram overlays show binding of the penta his antibody to the cells expressing the His6-human CD3 epsilon chain (His6- CD3) but not to the cells expressing the wild-type CD3 epsilon chain (WT-CD3).
- the lower Histogram overlays show binding of the I2C IgGI construct to the wild-type human CD3 epsilon chain but not to the His6-human CD3 epsilon chain.
- a single chain construct with irrelevant target specificity was used as negative control for binding to the MCSP D3 transfected CHO cells.
- the overlay of the histograms shows specific binding of the construct to human and macaque MCSP D3 and human and macaque CD3.
- Figure 60 FACS binding analysis of designated cross-species specific bispecific single chain constructs to CHO cells transfected with human CD33, the human CD3+ T cell line HPB-ALL, CHO cells transfected with macaque CD33 and macaque PBMC respectively.
- the FACS staining was performed as described in Example 36.2.
- the bold lines represent cells incubated with cell culture supernatant of transfected cells expressing the cross-species specific bispecific antibody constructs.
- the filled histograms reflect the negative controls. Supernatant of untransfected CHO cells was used as negative control.
- the overlay of the histograms shows specific binding of the construct to human and macaque CD33 and human and macaque CD3.
- the diagrams show results of chromium release assays measuring cytotoxic activity induced by designated cross-species specific CD33 specific single chain constructs redirected to the indicated target cell lines. Effector cells were also used as indicated.
- the assays are performed as described in Example 36.3.
- the diagrams clearly demonstrate for each construct the potent recruitment of cytotoxic activity of human and macaque effector cells against human and macaque CD33 transfected CHO cells, respectively.
- the infusion time for each bolus administration was 2 hours.
- Vertical arrows indicate the start of bolus infusions. Data points at the beginning of each bolus administration show the T cell counts immediately prior to start of bolus infusion.
- Detection was performed by a monoclonal anti Flag-Biotin-labeled antibody followed by peroxidase-conjugated Streptavidin.
- the ELISA was developed by an ABTS substrate solution.
- the OD values (y axis) were measured at 405 nm by an ELISA reader. Clone names are presented on the x axis.
- a single chain construct with irrelevant target specificity was used as negative control for binding to the CD44 and CD44delv6 transfected CHO cells.
- the overlay of the histograms shows specific binding of the construct to human and macaque CD44 and human and macaque CD3.
- the overlay of the histograms for each construct shows no specific binding to the human CD44delv6 transfected CHO cells.
- Cytotoxic activity induced by designated cross-species specific binding molecules as described in example 41 is induced by designated cross-species specific binding molecules as described in example 41.
- a reaction mix consisting of 4 ⁇ l of 5x superscript Il buffer, 0.2 ⁇ l of 0.1 M dithiothreitole, 0.8 ⁇ l of superscript Il (Invitrogen), 1.2 ⁇ l of desoxyribonucleoside triphosphates (25 ⁇ M), 0.8 ⁇ l of RNase Inhibitor (Roche) and 1.8 ⁇ l of DNase and RNase free water (Roth) was added.
- the reaction mix was incubated at room temperature for 10 minutes followed by incubation at 42 0 C for 50 minutes and at 90 0 C for 5 minutes.
- the reaction was cooled on ice before adding 0.8 ⁇ l of RNaseH (1 U/ ⁇ l, Roche) and incubated for 20 minutes at 37 0 C.
- the first-strand cDNAs from each species were subjected to separate 35-cycle polymerase chain reactions using Taq DNA polymerase (Sigma) and the following primer combination designed on database research: forward primer 5'- AGAGTTCTGGGCCTCTGC-3' (SEQ ID NO: 377); reverse primer 5'- CGGATGGGCTCATAGTCTG-3' (SEQ ID NO: 378);.
- the amplified 550 bp-bands were gel purified (Gel Extraction Kit, Qiagen) and sequenced (Sequiserve, Vaterstetten/Germany, see sequence listing).
- mice using the N-terminus of CD3epsilon separated from its native CD3-context by fusion to a heterologous soluble protein
- mice receive booster immunizations after 21 , 42 and optionally 63 days in the same way.
- Ten days after the first booster immunization blood samples were taken and antibody serum titer against 1-27 CD3-Fc fusion protein iwa tested by ELISA. Additionally, the titer against the CD3-positive human T cell line HPBaII was tested in flow cytometry according to standard protocols. Serum titers were significantly higher in immunized than in non-immunized animals.
- VK murine immunoglobulin
- VH Ig heavy chain variable region
- VH-VK scFv single chain variable fragment
- the E. coli cells containing the antibody library were transferred into SB-Carbenicillin (50 ug/mL) selection medium.
- the E. coli cells containing the antibody library wass then infected with an infectious dose of 10 12 particles of helper phage VCSM13 resulting in the production and secretion of filamentous M13 phage, wherein phage particle contains single stranded pComb3H5BHis-DNA encoding a murine scFv-fragment and displayed the corresponding scFv-protein as a translational fusion to phage coat protein III.
- This pool of phages displaying the antibody library was later used for the selection of antigen binding entities.
- Phage display based selection of CD3-specific binders The phage library carrying the cloned scFv-repertoire was harvested from the respective culture supernatant by PEG8000/NaCI precipitation and centrifugation. Approximately 10 11 to 10 12 scFv phage particles were resuspended in 0.4 ml of PBS/0.1% BSA and incubated with 10 5 to 10 7 Jurkat cells (a CD3-positive human T- cell line) for 1 hour on ice under slow agitation.
- E. coli XL1 Blue culture (OD600 > 0.5).
- Plasmid DNA corresponding to 4 and 5 rounds of panning was isolated from E. coli cultures after selection.
- VH-VL-DNA fragments were excised from the plasmids (Xhol-Spel). These fragments were cloned via the same restriction sites in the plasmid pComb3H5BFIag/His differing from the original pComb3H5BHis in that the expression construct (e.g. scFv) includes a Flag-tag (TGD YKDDDDK) between the scFv and the His6-tag and the additional phage proteins were deleted.
- the expression construct e.g. scFv
- TGD YKDDDDK Flag-tag
- each pool (different rounds of panning) of plasmid DNA was transformed into 100 ⁇ l heat shock competent E. coli TG1 or XLI blue and plated onto carbenicillin LB-agar. Single colonies were picked into 100 ul of LB carb (50 ug/ml).
- E. coli transformed with pComb3H5BHis containing a VL-and VH-segment produce soluble scFv in sufficient amounts after excision of the gene III fragment and induction with 1 mM IPTG. Due to a suitable signal sequence, the scFv-chain was exported into the periplasma where it folds into a functional conformation.
- coli TG 1 bacterial colonies from the transformation plates were picked for periplasmic small scale preparations and grown in SB-medium (e.g. 10 ml) supplemented with 20 mM MgCI2 and carbenicillin 50 ⁇ g/ml (and re-dissolved in PBS (e.g. 1 ml) after harvesting.
- SB-medium e.g. 10 ml
- carbenicillin 50 ⁇ g/ml and re-dissolved in PBS (e.g. 1 ml) after harvesting.
- PBS e.g. 1 ml
- the first amino acids 1-27 of the N-terminal sequence of the mature CD3 epsilon chain of the human T cell receptor complex (amino acid sequence: QDGNEEMGGITQTPYKVSISGTTVILT) were fused to the N-terminus of the transmembrane protein EpCAM so that the N-terminus was located at the outer cell surface. Additionally, a FLAG epitope was inserted between the N-terminal 1-27 CD3epsilon sequence and the EpCAM sequence. This fusion product was expressed in human embryonic kidney (HEK) and Chinese hamster ovary (CHO) cells.
- HEK human embryonic kidney
- CHO Chinese hamster ovary
- QDGNEEIGDTTQNPYKVSISGTTVTLT for Callithrix jacchus (CD3epsilon N- terminal amino acid sequence: QDGNEEMGDTTQNPYKVSISGTTVTLT) and for Saguinus oedipus (CD3epsilon N-terminal amino acid sequence: QDGNEEMGDTTQNPYKVSISGTTVTLT).
- a R-Phycoerythrin-conjugated affinity purified F(ab')2 fragment, goat anti-mouse IgG (Fc-gamma fragment specific), diluted 1 :100 in 50 ⁇ l PBS with 2% FCS (Dianova, Hamburg, FRG) was used.
- the samples were measured on a FACSscan (BD biosciences, Heidelberg, FRG).
- Binding was always confirmed by flowcytometry as described in the foregoing paragraph on primary T cells of man and different primates (e.g. saimiris ciureus, callithrix jacchus, saguinus oedipus).
- the VH region of the murine anti-CD3 scFv was aligned against human antibody germline amino acid sequences.
- the human antibody germline VH sequence was chosen which has the closest homology to the non-human VH and a direct alignment of the two amino acid sequences was performed.
- oligonucleotides were synthesized. These oligonucleotides incorporate at the differing positions the human residue with a probability of 75 % and the murine residue with a probability of 25 %. For one human VH e.g. six of these oligonucleotides had to be synthesized that overlap in a terminal stretch of approximately 20 nucleotides. To this end every second primer was an antisense primer. Restriction sites needed for later cloning within the oligonucleotides were deleted.
- These primers may have a length of 60 to 90 nucleotides, depending on the number of primers that were needed to span over the whole V sequence.
- These e.g. six primers were mixed in equal amounts (e.g. 1 ⁇ l of each primer (primer stocks 20 to 100 ⁇ M) to a 20 ⁇ l PCR reaction) and added to a PCR mix consisting of PCR buffer, nucleotides and Taq polymerase. This mix was incubated at 94 0 C for 3 minutes, 65 0 C for 1 minute, 62°C for 1 minute, 59 0 C for 1 minute, 56 0 C for 1 minute, 52 0 C for 1 minute, 50 0 C for 1 minute and at 72°C for 10 minutes in a PCR cycler.
- the product was run in an agarose gel electrophoresis and the product of a size from 200 to 400 isolated from the gel according to standard methods.
- This PCR product was then used as a template for a standard PCR reaction using primers that incorporate N-terminal and C-terminal suitable cloning restriction sites.
- the DNA fragment of the correct size (for a VH approximately 350 nucleotides) was isolated by agarose gel electrophoresis according to standard methods. In this way sufficient VH DNA fragment was amplified.
- This VH fragment was now a pool of VH fragments that have each one a different amount of human and murine residues at the respective differing framework positions (pool of humanized VH).
- VL region of the murine anti-CD3 scFv (pool of humanized VL).
- the pool of humanized VH was then combined with the pool of humanized VL in the phage display vector pComb3H5Bhis to form a library of functional scFvs from which - after display on filamentous phage - anti-CD3 binders were selected, screened, identified and confirmed as described above for the parental non-human (murine) anti-CD3 scFv.
- Single clones were then analyzed for favorable properties and amino acid sequence.
- scFvs which were closest in amino acid sequence homology to human germline V-segments are preferred particularly those wherein at least one CDR among CDR I and Il of VH and CDR I and Il of VLkappa or CDR I and Il of VLIambda shows more than 80% amino acid sequence identity to the closest respective CDR of all human germline V-segments.
- Anti-CD3 scFvs were converted into recombinant bispecific single chain antibodies as described in the following Examples 10 and 16 and further characterized.
- the coding sequence of the 1-27 N-terminal amino acids of the human CD3 epsilon chain fused to the hinge and Fc gamma region of human immunoglobulin IgGI as well as an 6 Histidine Tag were obtained by gene synthesis according to standard protocols (cDNA sequence and amino acid sequence of the recombinant fusion protein are listed under SEQ ID NOs 350 and 349).
- the gene synthesis fragment was designed as to contain first a Kozak site for eukaryotic expression of the construct, followed by an 19 amino acid immunoglobulin leader peptide, followed in frame by the coding sequence of the first 27 amino acids of the extracellular portion of the mature human CD3 epsilon chain, followed in frame by the coding sequence of the hinge region and Fc gamma portion of human IgGI , followed in frame by the coding sequence of a 6 Histidine tag and a stop codon ( Figure 1).
- the gene synthesis fragment was also designed as to introduce restriction sites at the beginning and at the end of the cDNA coding for the fusion protein.
- the introduced restriction sites, EcoRI at the 5' end and Sail at the 3' end, are utilized in the following cloning procedures.
- the gene synthesis fragment was cloned via EcoRI and Sail into a plasmid designated pEF-DHFR (pEF-DHFR is described in Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025) following standard protocols.
- a sequence verified plasmid was used for transfection in the FreeStyle 293 Expression System (Invitrogen GmbH, Düsseldorf, Germany) according to the manufacturers protocol. After 3 days cell culture supematants of the transfectants were harvested and tested for the presence of the recombinant construct in an ELISA assay.
- Goat anti-human IgG, Fc-gamma fragment specific antibody obtained from Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK was diluted in PBS to 5 ⁇ g/ml and coated with 100 ⁇ l per well onto a MaxiSorp 96-well ELISA plate (Nunc GmbH & Co. KG, Wiesbaden, Germany) over night at 4 0 C. Wells were washed with PBS with 0,05 % Tween 20 (PBS/Tween and blocked with 3 % BSA in PBS (bovine Albumin, fraction V, Sigma-Aldrich Chemie GmbH, Taufmün, Germany) for 60 minutes at room temperature (RT).
- Wells were washed with PBS with 0,05 % Tween 20 (PBS/Tween and blocked with PBS with 3 % BSA (bovine Albumin, fraction V, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) for 60 minutes at RT. Subsequently, wells were washed with PBS/Tween and incubated with supernatants of cells expressing the 1-27 CD3-Fc construct for 60 minutes at RT. Wells were washed with PBS/Tween and incubated with crude preparations of periplasmatically expressed cross-species specific single-chain antibodies as described above for 60 minutes at room temperature.
- BSA bovine Albumin, fraction V, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany
- CD3 epsilon was isolated from different non-chimpanzee primates (marmoset, tamarin, squirrel monkey) and swine.
- cDNA sequence and amino acid sequence of the recombinant fusion proteins are listed under SEQ ID NOs 351 to 360).
- the gene synthesis fragments were designed as to contain first a BsrGI site to allow fusion in correct reading frame with the coding sequence of a 19 amino acid immunoglobulin leader peptide already present in the target expression vector, which is followed in frame by the coding sequence of the N-terminal 1-27 amino acids of the extracellular portion of the mature CD3 epsilon chains, which is followed in frame by the coding sequence of a Flag tag and followed in frame by the coding sequence of the mature cynomolgus EpCAM transmembrane protein (Figure 4).
- the gene synthesis fragments were also designed to introduce a restriction site at the end of the cDNA coding for the fusion protein.
- the introduced restriction sites BsrGI at the 5' end and Sail at the 3' end, were utilized in the following cloning procedures.
- the gene synthesis fragments were then cloned via BsrGI and Sail into a derivative of the plasmid designated pEF DHFR (pEF-DHFR is described in Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025), which already contained the coding sequence of the 19 amino acid immunoglobulin leader peptide following standard protocols.
- Sequence verified plasmids were used to transiently transfect 293-HEK cells using the MATra-A Reagent (IBA GmbH, G ⁇ ttingen, Germany) and 12 ⁇ g of plasmid DNA for adherent 293-HEK cells in 175 ml cell culture flasks according to the manufacturers protocol. After 3 days of cell culture the transfectants were tested for cell surface expression of the recombinant transmembrane protein via an FACS assay according to standard protocols. For that purpose a number of 2,5x10 5 cells were incubated with the anti-Flag M2 antibody (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) at 5 ⁇ g/ml in PBS with 2% FCS.
- MATra-A Reagent IBA GmbH, G ⁇ ttingen, Germany
- Bound antibody was detected with an R-Phycoerythrin-conjugated affinity purified F(ab')2 fragment, goat anti-mouse IgG, Fc-gamma fragment specific 1 :100 in PBS with 2% FCS (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK). The samples were measured on a FACScalibur (BD biosciences, Heidelberg, Germany). Expression of the Flag tagged recombinant transmembrane fusion proteins consisting of cynomolgus EpCAM and the 1-27 N-terminal amino acids of the human, marmoset, tamarin, squirrel monkey and swine CD3 epsilon chain respectively on transfected cells was clearly detectable (Figure 5).
- Penta-His antibody As secondary antibody the Penta-His antibody (Qiagen GmbH, Hildesheim, Germany) was used at 5 ⁇ g/ml in 50 ⁇ l PBS with 2% FCS. The binding of the antibody was detected with an R-Phycoerythrin-conjugated affinity purified F(ab')2 fragment, goat anti-mouse IgG, Fc-gamma fragment specific, diluted 1 :100 in PBS with 2% FCS (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK). The samples were measured on a FACScalibur (BD biosciences, Heidelberg, Germany).
- the coding sequence of the human CD3 epsilon chain was obtained by gene synthesis according to standard protocols (cDNA sequence and amino acid sequence of the human CD3 epsilon chain are listed under SEQ ID NOs 362 and 361).
- the gene synthesis fragment was designed as to contain a Kozak site for eukaryotic expression of the construct and restriction sites at the beginning and the end of the cDNA coding for human CD3 epsilon.
- the introduced restriction sites EcoRI at the 5' end and Sail at the 3' end were utilized in the following cloning procedures.
- the gene synthesis fragment was then cloned via EcoRI and Sail into a plasmid designated pEF NEO following standard protocols.
- pEF NEO was derived of pEF DHFR (Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025) by replacing the cDNA of the DHFR with the cDNA of the neomycin resistance by conventional molecular cloning.
- a sequence verified plasmid was used to transfect the murine T cell line EL4 (ATCC No.
- TIB-39 cultivated in RPMI with stabilized L- glutamine supplemented with 10 % FCS, 1 % penicillin/streptomycin, 1% HEPES, 1% pyruvate, 1% non-essential amino acids (all Biochrom AG Berlin, Germany) at 37 0 C, 95 % humidity and 7 % CO2.
- Transfection was performed with the SuperFect Transfection Reagent (Qiagen GmbH, Hilden, Germany) and 2 ⁇ g of plasmid DNA according to the manufacturer's protocol. After 24 hours the cells were washed with PBS and cultivated again in the aforementioned cell culture medium with ⁇ OO ⁇ g/ml G418 for selection (PAA Laboratories GmbH, Pasching, Austria).
- H2C HLP In order to provide improved means of detection of binding of the cross-species specific single chain anti-CD3 antibodies H2C HLP, A2J HLP and E2M HLP were converted into IgGI antibodies with murine IgGI and human lambda constant regions.
- cDNA sequences coding for the heavy and light chains of respective IgG antibodies were obtained by gene synthesis according to standard protocols.
- the gene synthesis fragments for each specificity were designed as to contain first a Kozak site to allow eukaryotic expression of the construct, which is followed by an 19 amino acid immunoglobulin leader peptide (SEQ ID NOs 364 and 363), which is followed in frame by the coding sequence of the respective heavy chain variable region or respective light chain variable region, followed in frame by the coding sequence of the heavy chain constant region of murine IgGI (SEQ ID NOs 366 and 365) or the coding sequence of the human lambda light chain constant region (SEQ ID NO 368 and 367), respectively. Restriction sites were introduced at the beginning and the end of the cDNA coding for the fusion protein.
- Sequence verified plasmids were used for co-transfection of respective light and heavy chain constructs in the FreeStyle 293 Expression System (Invitrogen GmbH, Düsseldorf, Germany) according to the manufacturers protocol. After 3 days cell culture supernatants of the transfectants were harvested and used for the alanine-scanning experiment.
- Chimeric IgG antibodies as described in 2) and cross-species specific single chain antibodies specific for CD3 epsilon were tested in alanine-scanning experiment. Binding of the antibodies to the EL4 cell lines transfected with the alanine-mutant constructs of human CD3 epsilon as described in 3) was tested by FACS assay according to standard protocols. 2,5x10 5 cells of the respective transfectants were incubated with 50 ⁇ l of cell culture supernatant containing the chimeric IgG antibodies or with 50 ⁇ l of crude preparations of periplasmatically expressed single- chain antibodies.
- the anti-Flag M2 antibody (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) was used as secondary antibody at 5 ⁇ g/ml in 50 ⁇ l PBS with 2% FCS.
- a secondary antibody was not necessary.
- the binding of the antibody molecules was detected with an R-Phycoerythrin-conjugated affinity purified F(ab')2 fragment, goat anti-mouse IgG, Fc-gamma fragment specific, diluted 1 :100 in PBS with 2% FCS (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK).
- the EL4 cell lines transfected with the alanine-mutants for the amino acids tyrosine at position 15, valine at position 17, isoleucine at position 19, valine at position 24 or leucine at position 26 of the mature CD3 epsilon chain were not evaluated due to very low expression levels (data not shown).
- Binding of the cross-species specific single chain antibodies and the single chain antibodies in chimeric IgG format to the EL4 cell lines transfected with the alanine-mutants of human CD3 epsilon is shown in Figure 8 (A- D) as relative binding in arbitrary units with the geometric mean fluorescence values of the respective negative controls subtracted from all respective geometric mean fluorescence sample values.
- sample means the value in arbitrary units of binding depicting the degree of binding of a specific anti-CD3 antibody to a specific alanine-mutant as shown in Figure 8 (A-D), Sample means the geometric mean fluorescence value obtained for a specific anti-CD3 antibody assayed on a specific alanine-scanning transfectant, neg_Contr.
- UCHT-1 means the geometric mean fluorescence value obtained for the UCHT-1 antibody assayed on a specific alanine-mutant
- WT means the geometric mean fluorescence value obtained for a specific anti-CD3 antibody assayed on the wild-type transfectant
- x specifies the respective transfectant
- y specifies the respective anti-CD3 antibody
- wt specifies that the respective transfectant is the wild-type.
- the IgG antibody A2J HLP showed a pronounced loss of binding for the amino acids asparagine at position 4, threonine at position 23 and isoleucine at position 25 of the mature CD3 epsilon chain.
- a complete loss of binding of IgG antibody A2J HLP was observed for the amino acids glutamine at position 1 , aspartate at position 2, glycine at position 3 and glutamate at position 5 of the mature CD3 epsilon chain.
- IgG antibody E2M HLP showed a pronounced loss of binding for the amino acids asparagine at position 4, threonine at position 23 and isoleucine at position 25 of the mature CD3 epsilon chain.
- IgG antibody E2M HLP showed a complete loss of binding for the amino acids glutamine at position 1 , aspartate at position 2, glycine at position 3 and glutamate at position 5 of the mature CD3 epsilon chain.
- IgG antibody H2C HLP showed an intermediate loss of binding for the amino acid asparagine at position 4 of the mature CD3 epsilon chain and it showed a complete loss of binding for the amino acids glutamine at position 1 , aspartate at position 2, glycine at position 3 and glutamate at position 5 of the mature CD3 epsilon chain.
- Single chain antibody F12Q HLP showed an essentially complete loss of binding for the amino acids glutamine at position 1 , aspartate at position 2, glycine at position 3 of the mature CD3 epsilon chain and glutamate at position 5 of the mature CD3 epsilon chain.
- a cDNA fragment coding for the human CD3 epsilon chain with a N-terminal His6 tag was obtained by gene synthesis.
- the gene synthesis fragment was designed as to contain first a Kozak site for eukaryotic expression of the construct, which is followed in frame by the coding sequence of a 19 amino acid immunoglobulin leader peptide, which is followed in frame by the coding sequence of a His6 tag which is followed in frame by the coding sequence of the mature human CD3 epsilon chain (the cDNA and amino acid sequences of the construct are listed as SEQ ID NOs 380 and 379).
- the gene synthesis fragment was also designed as to contain restriction sites at the beginning and the end of the cDNA.
- 2,5x10 5 cells of the transfectants were incubated with 50 ⁇ l of cell culture supernatant containing the chimeric IgG antibody or 50 ⁇ l of the respective control antibodies at 5 ⁇ g/ml in PBS with 2% FCS.
- As negative control an appropriate isotype control and as positive control for expression of the constructs the CD3 specific antibody UCHT-1 were used respectively.
- the binding of the antibodies was detected with a R-Phycoerythrin-conjugated affinity purified F(ab')2 fragment, goat anti-mouse IgG, Fc-gamma fragment specific, diluted 1 :100 in PBS with 2% FCS (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK).
- a Surface Plasmon Resonance measurement was performed with a recombinant fusion protein consisting of the N-terminal amino acids 1-27 of the mature human CD3 epsilon chain fused to a Fc-part of human IgGI (1-27 CD3-Fc).
- a Biacore Carboxymethyl-Dextran CM5 chip (Biacore, Uppsala, Sweden) was installed on a Biacore 2000® system (Biacore, Uppsala, Sweden).
- One flow cell was activated by a ⁇ /-(3-Dimethylaminopropyl)-/V'-ethylcarbodiimide hydrochloride / N-Hydroxysuccinimide solution according to standard procedures.
- a solution of the fusion protein 1-27 CD3-Fc was added afterwards resulting in stable covalent linkage of the protein to the dextran layer of the Biacore chip. Unbound protein was removed by extensive washing followed by blocking of unreacted remaining NHS-activated carboxy groups by adding an ethanolamine solution. Success of protein coupling was confirmed by a higher signal measured as Response Units compared to the signal prior to coupling.
- a reference cell was prepared as described but without adding a protein solution.
- Purified bispecific antibody EGFR-21-63 LH x H2C HLP was extensively dialyzed against HBS-EP buffer (Biacore, Uppsala, Sweden) in a Slide-A-Lyzer® Mini Dialysis Unit (Pierce, Rockford-M, USA). Protein concentration after dialysis was determined by UV280 nm absorption resulting in a concentration of 43 ⁇ g/ml. The protein solution was transferred into a 96 well plate and serially diluted with HBS-EP buffer at a 1 :1 ratio to 10 further wells. Surface Plasmon Resonance Measurements were done by separately sampling all 11 wells. The flow cells were regenerated with acetate buffer between measurements to release bound protein.
- Binding signals of bispecific antibody molecules were obtained by subtraction of the signal of the reference cell from the signal of the measurement cell conjugated with the 1-27 CD3-Fc protein. Association and dissociation curves were measured as Response Units and recorded. The binding constants were calculated using the Biacore® curve fitting software based on the Langmuir model. The calculated binding constant KD over the first five concentrations was determined to be 1 ,52 x 10 "7 M. 7.2. Determination of CD3 binding constant by FACS measurement
- bispecific antibody molecule EGFR-21-63 LH x H2C HLP was used to set up a dilution row with a factor of 1 :1.5 and a starting concentration of 63.3 ⁇ g/ml.
- the bispecific antibody molecule was incubated at these different concentrations with 1.25 x 10 5 human PBMCs each for 1 hour a 4 0 C followed by two washing steps in PBS at 4°C.
- the detection of the bound bispecific antibody molecules was carried out by using a Penta-His antibody (Qiagen GmbH, Hildesheim, Germany) at 5 ⁇ g/ml in 50 ⁇ l PBS with 2% FCS. After incubation for 45 minutes at 4°C and two washing steps the binding of the Penta-His antibody was detected with an R-Phycoerythrin-conjugated affinity purified F(ab')2 fragment, goat anti-mouse IgG, Fc-gamma fragment specific, diluted 1 :100 in PBS with 2% FCS (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK).
- Flow cytometry was performed on a FACS-Canto Il apparatus, the FACS Diva software was used to acquire and analyze the data (Becton Dickinson biosciences, Heidelberg). FACS staining and measuring of the fluorescence intensity were performed as described in Current Protocols in Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober, Wiley-lnterscience, 2002). The acquired fluorescence intensity mean values were plotted as a function of the used bispecific antibody molecule concentration and analyzed by the biomathematical software Prism in a one side binding analysis (hyperbola).
- the software calculated the corresponding KD value that described the binding of a ligand (the bispecific antibody molecule) to a receptor (the CD3 positive PBMC subfraction) that follows the law of mass action.
- KD is the concentration of ligand required to reach half- maximal binding.
- the FACS staining was carried out in duplicates, the R 2 values were better than 0.95.
- the cell line positive for human EGFR, A431 (epidermoid carcinoma cell line, CRL- 1555, American Type Culture Collection, Rockville, MD) was used to obtain the total RNA that was isolated according to the instructions of the kit manual (Qiagen, RNeasy Mini Kit, Hilden, Germany). The obtained RNA was used for cDNA synthesis by random-primed reverse transcription.
- oligonucleotides were used: 5' EGFR AG Xbal 5'-GGTCTAGAGCATGCGACCCTCCGGGACGGCCGGG-S' 3' EGFR AG Sail 5'-TTTTAAGTCGACTCATGCTCCAATAAATTCACTGCT-S'
- the coding sequence was amplified by PCR (denaturation at 94 0 C for 5 min, annealing at 58°C for 1 min, elongation at 72°C for 2 min for the first cycle; denaturation at 94°C for 1 min, annealing at 58°C for 1 min, elongation at 72°C for 2 min for 30 cycles; terminal extension at 72 0 C for 5 min).
- the PCR product was subsequently digested with Xbal and Sail, ligated into the appropriately digested expression vector pEF-DHFR (Raum et al., Cancer Immunol. Immunother. 2001 ; 50: 141-150), and transformed into E.coli.
- the afore-mentioned procedures were carried out according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York (2001)).
- a clone with sequence-verified nucleotide sequence (SEQ ID 370, Amino acid sequence SEQ ID 369) was transfected into DHFR deficient CHO cells for eukaryotic expression of the construct.
- Eukaryotic protein expression in DHFR deficient CHO cells was performed as described by Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566. Gene amplification of the construct was induced by increasing concentrations of methothrexate (MTX) to a final concentration of up to 20 nM MTX. 9. Generation of CHO cells expressing the extracellular domain of cynomolgus EGFR
- the cDNA sequence of the extracellular domain of cynomolgus EGFR was obtained by a set of two PCRs on cynomolgus monkey colon cDNA (Cat#: C1534090-Cy-BC; obtained from BioCat GmbH, Heidelberg, Germany) using the following reaction conditions: 1 cycle at 94°C for 3 minutes followed by 35 cycles with 94°C for 1 minute, 53°C for 1 minute and 72°C for 2 minutes followed by a terminal cycle of 72°C for 3 minutes.
- the following primers were used:
- forward primer 5 1 - CGCTCTGCCCGGCGAGTCGGGC -3' reverse primer: 5'- CCGTCTTCCTCCATCTCATAGC -3'
- forward primer 5 1 - ACATCCGGAGGTGACAGATCACGGCTCGTGC -3' reverse primer: 5'- CAGGATATCCGAACGATGTGGCGCCTTCGC -3'
- PCRs generated two overlapping fragments (A: 1-869, B: 848-1923), which were isolated and sequenced according to standard protocols using the PCR primers, and thereby provided a 1923 bp portion of the cDNA sequence of cynomolgus EGFR from the third nucleotide of codon +1 of the mature protein to the 21 st codon of the transmembrane domain.
- a cDNA fragment was obtained by gene synthesis according to standard protocols (the cDNA and amino acid sequence of the construct is listed under SEQ ID Nos 372 and 371).
- the coding sequence for cynomolgus EGFR from amino acid +2 to +641 of the mature EGFR protein was fused into the coding sequence of human EGFR replacing the coding sequence of the amino acids +2 to +641.
- the gene synthesis fragment was also designed as to contain a Kozak site for eukaryotic expression of the construct and restriction sites at the beginning and the end of the cDNA coding for essentially the extracellular domain of cynomolgus EGFR fused to the transmembrane and intracellular domains of human EGFR.
- bispecific single chain antibody molecules each comprising a domain with a binding specificity cross-species specific for human and non-chimpanzee primate CD3 epsilon as well as a domain with a binding specificity cross-species specific for human and non-chimpanzee primate EGFR, were designed as set out in the following Table 1 :
- Table 1 Formats of anti-CD3 and anti-EGFR cross-species specific bispecific single chain antibody molecules
- variable light-chain (L) and variable heavy-chain (H) domains cross-species specific for human and cynomolgus EGFR were obtained by gene synthesis.
- the gene synthesis fragments were designed as to contain first a Kozak site for eukaryotic expression of the construct, followed by a 19 amino acid immunoglobulin leader peptide, followed in frame by the coding sequence of the respective bispecific single chain antibody molecule, followed in frame by the coding sequence of a 6 histidine tag and a stop codon.
- the gene synthesis fragment was also designed as to introduce suitable restriction sites at the beginning and at the end of the fragment. The introduced restriction sites were utilized in the following cloning procedures.
- the gene synthesis fragment was also designed as to introduce suitable N- and C-terminal restriction sites.
- the gene synthesis fragment was cloned via these restriction sites into a plasmid designated pEF-DHFR (pEF-DHFR is described in Griffin et al. Cancer Immunol lmmunother 50 (2001) 141-150) according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York (2001)).
- DHFR dihydrofolate reductase
- CHO Chinese hamster ovary
- the constructs were transfected stably or transiently into DHFR-deficient CHO-cells (ATCC No. CRL 9096) by electroporation or alternatively into HEK 293 (human embryonal kidney cells, ATCC Number: CRL-1573) in a transient manner according to standard protocols. 10.2. Expression and purification of the bispecific single chain antibody molecules
- the bispecific single chain antibody molecules were expressed in Chinese hamster ovary cells (CHO). Eukaryotic protein expression in DHFR deficient CHO cells was performed as described by Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566. Gene amplification of the constructs was induced by increasing final concentrations of MTX up to 20 nM. After two passages of stationary culture the cells were grown in roller bottles with nucleoside-free HyQ PF CHO liquid soy medium (with 4.0 mM L- Glutamine with 0.1 % Pluronic F - 68; HyClone) for 7 days before harvest. The cells were removed by centrifugation and the supernatant containing the expressed protein was stored at -20 0 C.
- CHO Chinese hamster ovary cells
- constructs were transiently expressed in HEK 293 cells. Transfection was performed with 293fectin reagent (Invitrogen, #12347-019) according to the manufacturer ' s protocol. Akta® Explorer System (GE Health Systems) and Unicorn® Software were used for chromatography. Immobilized metal affinity chromatography ("IMAC") was performed using a Fractogel EMD chelate® (Merck) which was loaded with ZnCI2 according to the protocol provided by the manufacturer. The column was equilibrated with buffer A (20 mM sodium phosphate buffer pH 7.2, 0.1 M NaCI) and the cell culture supernatant (500 ml) was applied to the column (10 ml) at a flow rate of 3 ml/min.
- buffer A (20 mM sodium phosphate buffer pH 7.2, 0.1 M NaCI
- the bispecific single chain antibody protein was analyzed in SDS PAGE under reducing conditions performed with pre-cast 4-12% Bis Tris gels (Invitrogen). Sample preparation and application were performed according to the protocol provided by the manufacturer. The molecular weight was determined with MultiMark protein standard (Invitrogen). The gel was stained with colloidal Coomassie (Invitrogen protocol). The purity of the isolated protein was >95% as determined by SDS-PAGE.
- the bispecific single chain antibody has a molecular weight of about 52 kDa under native conditions as determined by gel filtration in PBS. All constructs were purified according to this method.
- Western Blot was performed using an Optitran® BA-S83 membrane and the Invitrogen Blot Module according to the protocol provided by the manufacturer.
- the antibodies used were directed against the His Tag (Penta His, Qiagen) and Goat- anti-mouse Ig labeled with alkaline phosphatase (AP) (Sigma), and BCIP/NBT (Sigma) as substrate.
- His Tag Penta His, Qiagen
- AP alkaline phosphatase
- BCIP/NBT BCIP/NBT
- a Surface Plasmon Resonance measurement was performed with a recombinant fusion protein consisting of the N-terminal amino acids 1-27 of the human CD3 epsilon chain fused to a Fc-part of human IgGI (1-27 CD3-Fc).
- a Biacore Carboxymethyl- Dextran CM5 chip (Biacore, Uppsala, Sweden) was installed on a Biacore 2000® system (Biacore, Uppsala, Sweden).
- a flow cell was activated by a ⁇ /-(3- Dimethylaminopropyl)- ⁇ /'-ethylcarbodiimide hydrochloride / N-Hydroxysuccinimide solution according to standard procedures.
- a solution of the fusion protein 1-27 CD3- Fc was added afterwards resulting in stable covalent linkage of the protein to the dextran layer of the Biacore chip. Unbound protein was removed by extensive washing followed by blocking of remaining unreacted NHS-activated carboxy groups by adding an ethanolamine solution. Success of protein coupling was confirmed by detection of a higher signal measured as Response Units compared to the signal prior to coupling.
- a reference cell was prepared as described but without adding the protein solution. Purified bispecific single chain antibodies listed below were adjusted to 5 ⁇ g/ml with HBS-EP buffer (Biacore, Uppsala, Sweden) and transferred into a 96 well plate each at a volume of 150 ⁇ l.
- Binding signals of the bispecific single chain antibodies were obtained by subtraction of the signal of the reference cell from the signal of the measurement cell conjugated with the 1-27 CD3-Fc protein. Association and dissociation curves were measured as Response Units and recorded. The binding constants were calculated using the Biacore® curve-fitting software based on the Langmuir model. The calculated affinities for the tested fully cross-species specific bispecific single chain molecules to the N-terminal amino acids 1-27 of the human CD3 epsilon are given as KD values below and range from 2,54x10 "6 M to 2,49x10 '7 M. "LH” refers to an arrangement of variable domains in the order VL-VH. “HL” refers to an arrangement of variable domains in the order VH-VL. G4H, F70, A2J, E1 L, E2M, H1 E and F6A refer to different cross-species specific CD3 binding molecules.
- CHO cells transfected with human EGFR as described in Example 8 and human CD3 positive T cell leukemia cell line HPB-ALL were used to test the binding to human antigens.
- the binding reactivity to cynomolgus antigens was tested by using the generated cynomolgus EGFR transfectant described in Example 9 and a macaque T cell line 4119LnPx (kindly provided by Prof Fickenscher, Hygiene Institute, Virology, Er Weg-Nuernberg; published in Knappe A, et al., and Fickenscher H., Blood 2000, 95, 3256-61) 200.000 cells of the respective cell population were incubated for 30 min on ice with 50 ⁇ l of the purified protein of the cross-species specific bispecific antibody constructs (2 ⁇ g/ml). Alternatively, the cell culture supernatant of transiently produced proteins was used.
- the cells were washed twice in PBS and binding of the construct was detected with a murine Penta His antibody (Qiagen; diluted 1 :20 in 50 ⁇ l PBS with 2% FCS). After washing, bound anti His antibodies were detected with an Fc gamma-specific antibody (Dianova) conjugated to phycoerythrin, diluted 1 :100 in PBS with 2% FCS. Fresh culture medium was used as a negative control.
- Bioactivity of EGFR and CD3 cross-species specific bispecific single chain antibodies Bioactivity of the generated bispecific single chain antibodies was analyzed by chromium 51 ( 51 Cr) release in vitro cytotoxicity assays using the EGFR positive cell lines described in Examples 8 and 9. As effector cells stimulated human CD8 positive T cells or the macaque T cell line 4119LnPx were used, respectively. Generation of the stimulated CD8+ T cells was performed as follows: A Petri dish (145 mm diameter, Greiner) was pre-coated with a commercially available anti-CD3 specific antibody in a final concentration of 1 ⁇ g/ml for 1 hour at 37 0 C. Unbound protein was removed by one washing step with PBS.
- the fresh PBMCs were isolated from peripheral blood (30 - 50 ml human blood) by Ficoll gradient centrifugation according to standard protocols. 3 - 5 x 10 7 PBMCs were added to the precoated petri dish in 120 ml of RPMI 1640 / 10% FCS / IL-2 20 U/ml (Proleukin, Chiron) and stimulated for 2 days. At the third day the cells were collected, washed once with RPMI 1640. IL-2 was added to a final concentration of 20 U/ml and cultivated again for one day.
- CD8+ cytotoxic T lymphocytes (CTLs) were isolated by depletion of CD4+ T cells and CD56+ NK cells.
- Target cells were washed twice with PBS and labeled with 11 ,1 MBq 51 Cr in a final volume of 100 ⁇ l RPMI with 50% FCS for 45 minutes at 37 0 C. Subsequently the labeled target cells were washed 3 times with 5 ml RPMI and then used in the cytotoxicity assay.
- the assay was performed in a 96 well plate in a total volume of 250 ⁇ l supplemented RPMI (as above) with an E:T ratio of 10:1. 1 ⁇ g/ml of the cross- species specific bispecific single chain antibody molecules and 20 threefold dilutions thereof were applied. Alternatively cell culture supernatant of transiently produced proteins was serially diluted in 1 :2 steps.
- the assay time is 18 hours and cytotoxicity was measured as relative values of released chromium in the supernatant related to the difference of maximum lysis (addition of Triton-X) and spontaneous lysis (without effector cells). All measurements were done in quadruplicates. Measurement of chromium activity in the supernatants was performed with a Wizard 3" gammacounter (Perkin Elmer Life Sciences GmbH, KoIn, Germany). Analysis of the experimental data was performed with Prism 4 for Windows (version 4.02, GraphPad Software Inc., San Diego, California, USA). Sigmoidal dose response curves typically had R 2 values >0.90 as determined by the software. EC 50 values calculated by the analysis program were used for comparison of bioactivity.
- the coding sequence of the C-terminal, transmembrane and truncated extracellular domain of human MCSP was obtained by gene synthesis according to standard protocols (cDNA sequence and amino acid sequence of the recombinant construct for expression of the C-terminal, transmembrane and truncated extracellular domain of human MCSP (designated as human D3) are listed under SEQ ID NOs 374 and 373).
- the gene synthesis fragment was designed as to contain first a Kozak site to allow eukaryotic expression of the construct followed by the coding sequence of an 19 amino acid immunoglobulin leader peptide followed in frame by a FLAG tag, followed in frame by a sequence containing several restriction sites for cloning purposes and coding for a 9 amino acid artificial linker (SRTRSGSQL), followed in frame by the coding sequence of the C-terminal, transmembrane and truncated extracellular domain of human MCSP and a stop codon. Restriction sites were introduced at the beginning and at the end of the DNA fragment. The restriction sites EcoRI at the 5' end and Sail at the 3' end were used in the following cloning procedures.
- pEF-DHFR is described in Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025) following standard protocols.
- a sequence verified plasmid was used to transfect CHO/dhfr- cells (ATCC No. CRL 9096).
- Cells were cultivated in RPMI 1640 with stabilized glutamine, supplemented with 10% FCS, 1% penicillin/streptomycin (all obtained from Biochrom AG Berlin, Germany) and nucleosides from a stock solution of cell culture grade reagents (Sigma-Aldhch Chemie GmbH, Taufkirchen, Germany) to a final concentration of 10 ⁇ g/ml Adenosine, 10 ⁇ g/ml Deoxyadenosine and 10 ⁇ g/ml Thymidine, in an incubator at 37 °C, 95% humidity and 7% CO 2 .
- Transfection was performed using the PolyFect Transfection Reagent (Qiagen GmbH, Hilden, Germany) and 5 ⁇ g of plasmid DNA according to the manufacturer's protocol. After cultivation for 24 hours cells were washed once with PBS and cultivated again in RPMI 1640 with stabilized glutamine and 1% penicillin/streptomycin. Thus the cell culture medium did not contain nucleosides and thereby selection was applied on the transfected cells. Approximately 14 days after transfection the outgrowth of resistant cells was observed. After an additional 7 to 14 days the transfectants were tested for expression of the construct by FACS analysis.
- 2,5x10 5 cells were incubated with 50 ⁇ l of an anti-Flag-M2 antibody (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) diluted to 5 ⁇ g/ml in PBS with 2% FCS.
- the binding of the antibody was detected with a R-Phycoerythrin-conjugated affinity purified F(ab')2 fragment, goat anti-mouse IgG, Fc-gamma fragment specific diluted 1 :100 in PBS with 2% FCS (ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK).
- the samples were measured on a FACScalibur (BD biosciences, Heidelberg, Germany).
- the cDNA sequence of the C-terminal, transmembrane and truncated extracellular domains of macaque MCSP (designated as macaque D3) was obtained by a set of three PCRs on macaque skin cDNA (Cat No. C1534218-Cy-BC; BioCat GmbH, Heidelberg, Germany) using the following reaction conditions: 1 cycle at 94°C,3 min., 40 cycles with 94°C for 0,5 min., 52°C for 0,5 min. and 72°C for 1 ,75 min., terminal cycle of 72°C for 3 min..
- forward primer 5'-GATCTGGTCTACACCATCGAGC-3 1 reverse primer: 5'-GGAGCTGCTGCTGGCTCAGTGAGG-3 1 forward primer: 5'- TTCCAGCTGAGCATGTCTGATGG-3' reverse primer: 5'- CGATCAGCATCTGGGCCCAGG-3' forward primer: 5'- GTGGAGCAGTTCACTCAGCAGGACC-3' reverse primer: 5'- GCCTTCACACCCAGTACTGGCC-3'
- PCRs generated three overlapping fragments (A: 1-1329, B: 1229-2428, C: 1782-2547) which were isolated and sequenced according to standard protocols using the PCR primers and thereby provided a 2547 bp portion of the cDNA sequence of macaque MCSP (the cDNA sequence and amino acid sequence of this portion of macaque MCSP are listed under SEQ ID NOs 376 and 375) from 74 bp upstream of the coding sequence of the C-terminal domain to 121 bp downstream of the stop codon.
- primers for this PCR were designed to introduce restriction sites at the beginning and at the end of the cDNA fragment coding for the C-terminal, transmembrane and truncated extracellular domains of macaque MCSP.
- PCR fragment was then cloned via Mfel and Sail into a Bluescript plasmid containing the EcoRI/Mfel fragment of the aforementioned plasmid pEF- DHFR (pEF-DHFR is described in Griffin et al. Cancer Immunol lmmunother 50 (2001) 141-150) by replacing the C-terminal, transmembrane and truncated extracellular domains of human MCSP.
- the gene synthesis fragment contained the coding sequences of the immunoglobulin leader peptide and the Flag tag as well as the artificial linker (SRTRSGSQL) in frame to the 5' end of the cDNA fragment coding for the C-terminal, transmembrane and truncated extracellular domains of macaque MCSP.
- This vector was used to transfect CHO/dhfr- cells (ATCC No. CRL 9096).
- Cells were cultivated in RPMI 1640 with stabilized glutamine supplemented with 10% FCS, 1% penicillin/streptomycin (all from Biochrom AG Berlin, Germany) and nucleosides from a stock solution of cell culture grade reagents (Sigma-Aldrich Chemie GmbH, Taufmün, Germany) to a final concentration of 10 ⁇ g/ml Adenosine, 10 ⁇ g/ml Deoxyadenosine and 10 ⁇ g/ml Thymidine, in an incubator at 37 0 C, 95% humidity and 7% CO2. Transfection was performed with PolyFect Transfection Reagent (Qiagen GmbH, Hilden, Germany) and 5 ⁇ g of plasmid DNA according to the manufacturer's protocol.
- Bound antibody was detected with a R-Phycoerythrin- conjugated affinity purified F(ab')2 fragment, goat anti-mouse IgG, Fc-gamma fragment specific, diluted 1 :100 in PBS with 2% FCS (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK). Samples were measured on a FACScalibur (BD biosciences, Heidelberg, Germany).
- Bispecific single chain antibody molecules each comprising a binding domain cross- species specific for human and non-chimpanzee primate CD3 epsilon as well as a binding domain cross-species-specific for human and non-chimpanzee primate MCSP, are designed as set out in the following Table 2:
- Table 2 Formats of MCSP and CD3 cross-species specific bispecific single chain antibodies
- variable heavy-chain (VH) and variable light-chain (VL) domains cross-species specific for human and macaque MCSP D3 and the VH and VL domains cross-species specific for human and macaque CD3 were obtained by gene synthesis.
- the gene synthesis fragments were designed as to contain first a Kozak site for eukaryotic expression of the construct, followed by a 19 amino acid immunoglobulin leader peptide, followed in frame by the coding sequence of the respective bispecific single chain antibody molecule, followed in frame by the coding sequence of a histidine 6 -tag and a stop codon.
- the gene synthesis fragment was also designed as to introduce suitable N- and C-terminal restriction sites.
- pEF-DHFR plasmid designated pEF-DHFR
- the constructs were transfected stably or transiently into DHFR-deficient CHO-cells (ATCC No. CRL 9096) by electroporation or alternatively into HEK 293 (human embryonal kidney cells, ATCC Number: CRL-1573) in a transient manner according to standard protocols.
- Eukaryotic protein expression in DHFR deficient CHO cells was performed as described by Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566. Gene amplification of the constructs was induced by addition of increasing concentrations of methothrexate (MTX) up to final concentrations of 20 nM MTX. After two passages of stationary culture the cells were grown in roller bottles with nucleoside-free HyQ PF CHO liquid soy medium (with 4.0 mM L-Glutamine with 0.1% Pluronic F - 68; HyClone) for 7 days before harvest. The cells were removed by centrifugation and the supernatant containing the expressed protein is stored at -20 0 C.
- MTX methothrexate
- IMAC Immobilized metal affinity chromatography
- Bound protein was eluted using a two step gradient of buffer B (20 mM sodium phosphate buffer pH 7.2, 0.1 M NaCI, 0.5 M Imidazole) according to the following: Step 1 : 20% buffer B in 6 column volumes Step 2: 100% buffer B in 6 column volumes
- Eluted protein fractions from step 2 were pooled for further purification. All chemicals are of research grade and purchased from Sigma (Deisenhofen) or Merck (Darmstadt).
- the bispecific single chain antibody protein was analyzed in SDS PAGE under reducing conditions performed with pre-cast 4-12% Bis Tris gels (Invitrogen). Sample preparation and application were performed according to the protocol provided by the manufacturer. The molecular weight was determined with MultiMark protein standard (Invitrogen). The gel was stained with colloidal Coomassie (Invitrogen protocol). The purity of the isolated protein is >95% as determined by SDS-PAGE.
- the bispecific single chain antibody has a molecular weight of about 52 kDa under native conditions as determined by gel filtration in phosphate buffered saline (PBS). All constructs were purified according to this method.
- 4 x 105 cells per construct were cultivated in 3 ml RPMI 1640 all medium with stabilized glutamine supplemented with 10% fetal calf serum, 1% penicillin/streptomycin and nucleosides from a stock solution of cell culture grade reagents (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) to a final concentration of 10 ⁇ g/ml Adenosine, 10 ⁇ g/ml Deoxyadenosine and 10 ⁇ g/ml Thymidine, in an incubator at 37 0 C, 95% humidity and 7% CO2 one day before transfection.
- Bioactivity of MCSP and CD3 cross-species specific bispecific single chain antibodies Bioactivity of the generated bispecific single chain antibodies was analyzed by chromium 51 ( 51 Cr) release in vitro cytotoxicity assays using the MCSP D3 positive cell lines described in Examples 14 and 15.
- As effector cells stimulated human CD4/CD56 depleted PBMC, stimulated human PBMC or the macaque T cell line 4119LnPx are used as specified in the respective figures.
- Generation of the stimulated CD4/CD56 depleted PBMC was performed as follows: Coating of a Petri dish (145 mm diameter, Greiner bio-one GmbH, Kremsm ⁇ nster) was carried out with a commercially available anti-CD3 specific antibody (e.g.
- PBMC peripheral blood (30 - 50 ml human blood) by Ficoll gradient centhfugation according to standard protocols. 3 - 5 x 10 7 PBMC were added to the precoated petri dish in 120 ml of RPMI 1640 with stabilized glutamine / 10% FCS / IL-2 20 U/ml (Proleukin, Chiron) and stimulated for 2 days. On the third day the cells were collected and washed once with RPMI 1640.
- IL-2 was added to a final concentration of 20 U/ml and the cells were cultivated again for one day in the same cell culture medium as above.
- CD8+ cytotoxic T lymphocytes CTLs
- Target cells were washed twice with PBS and labelled with 11.1 MBq 51 Cr in a final volume of 100 ⁇ l RPMI with 50% FCS for 45 minutes at 37°C. Subsequently the labelled target cells were washed 3 times with 5 ml RPMI and then used in the cytotoxicity assay.
- the assay was performed in a 96 well plate in a total volume of 250 ⁇ l supplemented RPMI (as above) with an E:T ratio 10:1. 1 ⁇ g/ml of the cross- species specific bispecific single chain antibody molecules and 20 threefold dilutions thereof were applied. If using supernatant containing the cross-species specific bispecific single chain antibody molecules, 21 two- and 20 threefold dilutions thereof were applied for the macaque and the human cytotoxicity assay, respectively.
- the assay time was 18 hours and cytotoxicity was measured as relative values of released chromium in the supernatant related to the difference of maximum lysis (addition of Triton-X) and spontaneous lysis (without effector cells).
- Bioactivity was studied in a chromium 51 ( 51 Cr) release in vitro cytotoxicity assay using a MCSP positive CHO cell line (expressing MCSP as cloned according to example 14 or 15) as target and stimulated human CD8 positive T cells as effector cells.
- EC 50 values calculated by the analysis program as described above were used for comparison of bioactivity of bispecific single chain antibodies incubated with 50% human plasma for 24 hours at 37°C and 4 0 C respectively with bispecific single chain antibodies without addition of plasma or mixed with the same amount of plasma immediately prior to the assay.
- B-NHL B-cell Non-Hodgkin-Lymphoma
- a conventional CD19xCD3 binding molecules is a CD3 binding molecule of the bispecific tandem scFv format (Loffler (2000, Blood, Volume 95, Number 6) or WO 99/54440). It consists of two different binding portions directed at (i) CD19 on the surface of normal and malignant human B cells and (ii) CD3 on human T cells. By crosslinking CD3 on T cells with CD19 on B cells, this construct triggers the redirected lysis of normal and malignant B cells by the cytotoxic activity of T cells.
- the CD3 epitope recognized by such a conventional CD3 binding molecule is localized on the CD3 epsilon chain, where it only takes the correct conformation if it is embedded within the rest of the epsilon chain and held in the right position by heterodimerization of the epsilon chain with either the CD3 gamma or delta chain. Interaction of this highly context dependent epitope with a conventional CD3 binding molecule (see e.g.
- monovalent conventional CD3 binding molecules may induce some T cell reactions when infused into humans even in those cases where no circulating target cells are available for CD3 crosslinking.
- An important T cell reaction to the intravenous infusion of monovalent conventional CD19xCD3 binding molecule into B-NHL patients who have essentially no circulating CD19-positive B cells is the redistribution of T cells after start of treatment.
- Measurable disease at least one lesion > 1.5 cm
- patients received conventional CD19xCD3 binding molecule by continuous intravenous infusion with a portable minipump system over four weeks at constant flow rate (i.e. dose level).
- dose level i.e. dose level
- Patients were hospitalized during the first two weeks of treatment before they were released from the hospital and continued treatment at home.
- Patients without evidence of disease progression after four weeks were offered to continue treatment for further four weeks. So far six different dose levels were tested without reaching a maximum tolerated dose (MTD): 0.5, 1.5, 5, 15, 30 and 60 ⁇ g/m 2 /24h.
- MTD maximum tolerated dose
- PBMC peripheral blood mononuclear cells isolation was performed by an adapted FicollTM gradient separation protocol. Blood was transferred at room temperature into 10 ml LeucosepTM tubes (Greiner) pre-loaded with 3 ml BiocollTM solution (Biochrom). Centrifugation was carried out in a swing-out rotor for 15 min at 1700xg and 22°C without deceleration. The PBMC above the BiocollTM layer were isolated, washed once with FACS buffer (PBS / 2% FBS [Foetal Bovine Serum; Biochrom]), centrifuged and resuspended in FACS buffer.
- FACS buffer PBS / 2% FBS [Foetal Bovine Serum; Biochrom]
- Monoclonal antibodies were obtained from Invitrogen ( 1 Cat. No. MHCD1301 , 2 Cat. No. MHCD1401), Dako ( 5 Cat. No. C7224) or Becton Dickinson ( 3 Cat. No. 555516, 4 Cat. No. 345766) used according to the manufacturers " recommendations.
- 5x10 5 - 1x10 6 cells were stained with the following antibody combination: anti-CD13 1 / anti- CD14 2 (FITC) x anti-CD56 3 (PE) x anti-CD3 4 (PerCP) x anti-CD19 5 (APC). Cells were pelleted in V-shaped 96 well multititer plates (Greiner) and the supernatant was removed.
- T cell redistribution during the starting phase of conventional CD19xCD3 binding molecule e.g. disclosed in WO 99/54440
- a representative example of T cell redistribution during the starting phase of CD19xCD3 binding molecule treatment in a patient with a significant number of circulating CD19-positive B cells is shown in Fig. 26. In both cases (i.e. essentially no or many circulating B cells) circulating T cell counts rapidly decrease upon treatment start.
- T cells tend to return into the circulating blood very early, while the return of T cells into the circulating blood of those patients who have a significant number of circulating B cells at treatment start is usually delayed until these circulating B cells are depleted.
- the T cell redistribution patterns mainly differ in the kinetics of T cell reappearance in the circulating blood.
- SPD deviations from baseline between +50% and -25% were regarded as stable disease (SD).
- Patient demographics, doses received and clinical outcome in 34 patients are summarized in Table 4.
- Clinical anti-tumor activity of the CD19xCD3 binding molecule was clearly dose dependent: Consistent depletion of circulating CD19- positive B (lymphoma) cell from peripheral blood was observed from 5 ⁇ g/m 2 /24h onwards. At 15 ⁇ g/m 2 /24h and 30 ⁇ g/m 2 /24h first objective clinical responses (PRs and CRs) were recorded as well as cases of partial and complete elimination of B lymphoma cells from infiltrated bone marrow.
- PRs and CRs first objective clinical responses
- CD19xCD3 binding molecule-related adverse events usually were transient and fully reversible.
- the corresponding T cell redistribution pattern shows that circulating T cell counts rapidly decreased upon start of infusion at 5 ⁇ g/m 2 /24h followed by early reappearance of T cells in the circulating blood essentially without circulating CD19-positive B cells.
- the peripheral T cell counts had fully recovered when the CD19xCD3 binding molecule dose was increased after 24h from 5 to 15 ⁇ g/m 2 /24h. Therefore the dose step could trigger a second episode of T cell redistribution as shown in Fig. 24 A.
- This repeated T cell redistribution was related with CNS side effects (lead symptoms: confusion and disorientation) in this patient, which led to the stop of infusion.
- Endothelial cell activation by attached T cells can have procoagulatory effects (Monaco et al. J Leukoc Biol 71 (2002) 659- 668) with possible disturbances in blood flow (including cerebral blood flow) particularly with regard to capillary microcirculation.
- CNS adverse events related to T cell redistribution in patients essentially without circulating target cells can be the consequence of capillary leak and/or disturbances in capillary microcirculation through adherence of T cells to endothelial cells.
- the endothelial stress caused by one episode of T cell redistribution is tolerated by the majority of patients, while the enhanced endothelial stress caused by repeated T cell redistribution frequently causes CNS adverse events. More than one episode of T cell redistribution may be less risky only in patients who have low baseline counts of circulating T cells. However, also the limited endothelial stress caused by one episode of T cell redistribution can cause CNS adverse events in rare cases of increased susceptibility for such events as observed in 1 out of 21 patients in the bolus infusion trials with the CD19xCD3 binding molecule.
- the transient increase of T cell adhesiveness to the endothelial cells in patients who have essentially no circulating target cells can be explained as T cell reaction to the monovalent interaction of a conventional CD3 binding molecule, like the CD19xCD3 binding molecule (e.g. WO 99/54440), to its context dependent epitope on CD3 epsilon resulting in an allosteric change in the conformation of CD3 followed by the recruitment of Nck2 to the cytoplasmic domain of CD3 epsilon as described above.
- a conventional CD3 binding molecule like the CD19xCD3 binding molecule (e.g. WO 99/54440)
- Nck2 is directly linked to integrins via PINCH and ILK (Fig 28)
- recruitment of Nck2 to the cytoplasmic domain of CD3 epsilon following an allosteric change in the conformation of CD3 through binding of a conventional CD3 binding molecule, like the CD19xCD3 binding molecule, to its context dependent epitope on CD3 epsilon can increase the adhesiveness of T cells to endothelial cells by transiently switching integrins on the T cell surface into their more adhesive isoform via inside-out-signalling.
- AE adverse event
- AP alkaline phosphatase
- LDH lactate dehydrogenase
- CRP C-reactive protein
- ALT alanine transaminase
- AST aspartate transaminase
- GGT gamma-glutamyl transferase
- CD3 binding molecules of the invention capable of binding to a context-dependent epitope, though functional, lead to the undesired effect of T cell redistribution in patients causing CNS adverse events.
- binding molecules of the present invention by binding to the context-independent N-terminal 1-27 amino acids of the CD3 epsilon chain, do not lead to such T cell redistribution effects.
- the CD3 binding molecules of the invention are associated with a better safety profile compared to conventional CD3 binding molecules. 21.
- Bispecific CD3 binding molecules of the invention inducing T cell mediated target cell lysis by recognizing a surface target antigen deplete target antigen positive cells in vivo
- a bispecific CD3 binding molecule of the invention recognizing CD33 as target antigen depletes CD33-positive circulating monocytes from the peripheral blood of cynomolgus monkeys
- CD33-AF5 VH-VL x I2C VH-VL (amino acid sequence: SEQ ID NO.415) was produced by expression in CHO cells using the coding nucleotide sequence SEQ ID NO. 416.
- the coding sequences of (i) an N-terminal immunoglobulin heavy chain leader comprising a start codon embedded within a Kozak consensus sequence and (ii) a C-terminal His 6 -tag followed by a stop codon were both attached in frame to the nucleotide sequence SEQ ID NO 416 prior to insertion of the resulting DNA-fragment as obtained by gene synthesis into the multiple cloning site of the expression vector pEF-DHFR (Raum et al.
- Protein purification from the harvest of 3 production runs was based on IMAC affinity chromatography targeting the C-terminal His6-tag of CD33- AF5 VH-VL x I2C VH-VL followed by preparative size exclusion chromatography (SEC).
- SEC preparative size exclusion chromatography
- the total yield of final endotoxin-free test material was 60 mg.
- the analytical SEC-profile of CD33-AF5 VH-VL x I2C VH-VL for use in cynomolgus monkeys revealed that the test material almost exclusively consisted of monomer.
- the potency of the test material was measured in a cytotoxicity assay as described in example 23.5 using CHO cells transfected with cynomolgus CD33 as target cells and the macaque T cell line 4119LnPx as source of effector cells (FIG. 29).
- the concentration of CD33-AF5 VH-VL x I2C VH-VL required for half-maximal target cell lysis by the effector T cells (EC50) was determined to be 2.7 ng/ml. Young (approx.
- CD19-positive target B cells from the peripheral blood had turned out as a valid surrogate for the general clinical efficacy of the conventional CD3 binding molecule (CD19xCD3 as provided in WO99/54440) in patients with CD19-positive B- cell malignomas like B-NHL.
- depletion of circulating CD33-positive monocytes from the peripheral blood is regarded as a valid surrogate of the general clinical efficacy of CD33-directed bispecific CD3 binding molecules of the invention like CD33-AF5 VH-VL x I2C VH-VL in patients with CD33-positive myeloid malignomas like AML (acute myeloid leukemia).
- Continuous infusion was carried out according to the Swivel method as follows: The monkeys are catheterized via the vena femoralis into the vena cava caudalis using a vein catheter. The catheter is tunneled subcutaneously to the dorsal shoulder region and exteriorized at the caudal scapula. Then a tube is passed through a jacket and a protection spring. The jacket is fastened around the animal and the catheter, via the tube, is connected to an infusion pump. Administration solution (1.25 M lysine, 0.1 % tween 80, pH 7) without test material was infused continuously at 48 ml/24h for 7 days prior to treatment start to allow acclimatization of the animals to the infusion conditions.
- Treatment was started by adding CD33-AF5 VH-VL x I2C VH-VL test material to the administration solution at the amount required for each individual dose level to be tested (i.e. flow rate of CD33-AF5 VH-VL x I2C VH-VL).
- the infusion reservoir was changed every day throughout the whole acclimatization and treatment phase. Planned treatment duration was 7 days except for the 120 ⁇ g/m 2 /24h dose level, where animals received 14 days of treatment.
- Time courses of absolute counts in circulating T cells and CD33-positive monocytes were determined by 4- or 3-colour FACS analysis, respectively: Collection of blood samples and routine analysis
- Blood samples (1 ml) were obtained before and 0.75, 2, 6, 12, 24, 30, 48, 72 hours after start of continuous infusion with MCSP-G4 VH-VL x I2C VH-VL as well as after 7 and 14 days (and after 9 days at the 120 ⁇ g/m 2 /24h dose level) of treatment using EDTA-containing VacutainerTM tubes (Becton Dickinson) which were shipped for analysis at 4°C. In some cases slight variations of these time points occurred for operational reasons. FACS analysis of lymphocyte subpopulations was performed within 24 - 48 h after blood sample collection. Absolute numbers of leukocyte subpopulations in the blood samples were determined through differential blood analysis in a routine veterinary lab.
- PBMC isolation was performed by an adapted FicollTM gradient separation protocol. Blood was transferred at room temperature into 5 ml FalconTM tubes pre-loaded with 1 ml BiocollTM solution (Biochrom). Centhfugation was carried out in a swing-out rotor for 15 min at 1700xg and 22°C without deceleration. The PBMC above the BiocollTM layer were isolated, washed once with FACS buffer (PBS / 2% FBS [Foetal Bovine Serum; Biochrom]), centrifuged and resuspended in FACS buffer.
- FACS buffer PBS / 2% FBS [Foetal Bovine Serum; Biochrom]
- Monoclonal antibodies reactive with cynomolgus antigens were obtained from Becton Dickinson ( 1 Cat. No. 345784, 2 Cat. No. 556647, 3 Cat. No. 552851 , 6 Cat. No. 557710), Beckman Coulter ( 4 Cat. No. IM2470) and Miltenyi ( 5 Cat. No. 130-091-732) and used according to the manufacturers' recommendations.
- 5x10 5 - 1x10 6 cells were stained with the following antibody combinations: anti-CD14 1 (FITC) x anti- CD56 2 (PE) x anti-CD3 3 (PerCP) x anti-CD19 4 (APC) and anti-CD14 1 (FITC) x anti- CD33 5 (PE) x anti-CD16 6 (Alexa Fluor 647TM).
- Cells were pelleted in V-shaped 96 well multititer plates (Greiner) and the supernatant was removed. Cell pellets were resuspended in a total volume of 100 ⁇ l containing the specific antibodies diluted in FACS buffer. Incubation was carried out in the dark for 30 min at 4°C. Subsequently, samples were washed twice with FACS buffer and cell pellets were resuspended in FACS buffer for flowcytometric analysis. Flowcytometric detection of stained lymphocytes by FACS
- Absolute numbers of CD33-positive monocytes were calculated by multiplying the monocyte counts from the differential blood analysis with the corresponding ratios of CD33-positive monocytes (CD33 + , CD14 + ) to all monocytes (CD14 + ) as determined by FACS. The percentage compared to baseline (i.e. 100%) of absolute circulating CD33- positive monocyte counts at the end of treatment with CD33-AF5 VH-VL x I2C VH-VL in 4 cohorts of 2 cynomolgus monkeys with inter-cohort dose escalation from 30 over 60 and 240 to 1000 ⁇ g/m 2 /24h are shown in FIG 30 A.
- CD3 binding molecules of the invention directed at essentially context independent CD3 epitopes by inducing less redistribution of circulating T cells in the absence of circulating target cells reduce the risk of adverse events related to the initiation of treatment
- MCSP-G4 VH-VL x I2C VH-VL (amino acid sequence: SEQ ID NO. 313) was produced by expression in CHO cells using the coding nucleotide sequence SEQ ID NO. 314.
- the coding sequences of (i) an N-terminal immunoglobulin heavy chain leader comprising a start codon embedded within a Kozak consensus sequence and (ii) a C-terminal His6-tag followed by a stop codon were both attached in frame to the nucleotide sequence SEQ ID NO. 314 prior to insertion of the resulting DNA- fragment as obtained by gene synthesis into the multiple cloning site of the expression vector pEF-DHFR (Raum et al.
- Protein purification from the harvest was based on IMAC affinity chromatography targeting the C-terminal His6-tag of MCSP-G4 VH-VL x I2C VH-VL followed by preparative size exclusion chromatography (SEC).
- SEC preparative size exclusion chromatography
- the total yield of final endotoxin-free test material was 40 mg.
- the test material consisted of 70 % monomer, 30% dimer and a small contamination of higher multimer.
- the potency of the test material was measured in a cytotoxicity assay as described in example 18 using CHO cells transfected with cynomolgus MCSP as target cells and the macaque T cell line 4119LnPx as source of effector cells (FIG 31).
- the concentration of MCSP-G4 VH-VL x I2C VH-VL required for half-maximal target cell lysis by the effector T cells was determined to be 1.9 ng/ml.
- Young (approx. 3 years old) adult cynomolgus monkeys (Macaca fasciculahs) were treated by continuous intravenous infusion of CD3 binding molecule MCSP-G4 VH- VL x I2C VH-VL at different flow-rates (i.e. dose levels) to study redistribution of circulating T cells following initiation of treatment in the absence of circulating target cells.
- the CD3 binding molecule MCSP-G4 VH-VL x I2C VH-VL can recognize both cynomolgus MCSP and cynomolgus CD3, there are no circulating blood cells expressing MCSP. Therefore, the only interaction possible in the circulating blood is binding of the CD3-specific arm of MCSP-G4 VH-VL x I2C VH-VL to CD3 on T cells. This situation is equivalent to the treatment with the conventional CD3 binding molecule (CD19xCD3 binding molecule specific for CD19 on B cells and CD3 on T cells) of those B-NHL patients, who have no circulating CD19-positive target B cells as described in example 20.
- Administration solution (1.25 M lysine, 0.1% tween 80, pH 7) without test material was infused continuously at 48 ml/24h for 7 days prior to treatment start to allow acclimatization of the animals to the infusion conditions.
- Treatment was started by adding MCSP-G4 VH-VL x I2C VH-VL test material to the administration solution at the amount required for each individual dose level to be tested (i.e. flow rate of MCSP-G4 VH-VL x I2C VH-VL).
- the infusion reservoir was changed every day throughout the whole acclimatization and treatment phase. Treatment duration was 7 days.
- Time courses of absolute T-cell counts in peripheral blood were determined by four color FACS analysis as follows: Collection of blood samples and routine analysis Blood samples (1 ml) were obtained before and 0.75, 2, 6, 12, 24, 30, 48, 72 hours after start of continuous infusion with MCSP-G4 VH-VL x I2C VH-VL as well as after 7 days of treatment using EDTA-containing VacutainerTM tubes (Becton Dickinson) which were shipped for analysis at 4°C. In some cases slight variations of these time points occurred for operational reasons. FACS analysis of lymphocyte subpopulations was performed within 24 - 48 h after blood sample collection. Absolute numbers of leukocyte subpopulations in the blood samples were determined through differential blood analysis in a routine veterinary lab.
- PBMC isolation was performed by an adapted FicollTM gradient separation protocol. Blood was transferred at room temperature into 5 ml FalconTM tubes pre-loaded with 1 ml BiocollTM solution (Biochrom). Centrifugation was carried out in a swing-out rotor for 15 min at 1700xg and 22°C without deceleration. The PBMC above the BiocollTM layer were isolated, washed once with FACS buffer (PBS / 2% FBS [Foetal Bovine Serum; Biochrom]), centrifuged and resuspended in FACS buffer.
- FACS buffer PBS / 2% FBS [Foetal Bovine Serum; Biochrom]
- Centrifugation during all wash steps was carried out in a swing-out rotor for 4 min at 800xg and 4 0 C. If necessary, lysis of erythrocytes was performed by incubating the isolated PBMC in 3 ml erythrocyte lysis buffer (8.29 g NH 4 CI, 1.00 g KHCO 3 , 0.037 g EDTA, ad 1.0 I H 2 O bid est, pH 7.5) for 5 min at room temperature followed by a washing step with FACS buffer.
- T cell redistribution during the starting phase of treatment with MCSP-G4 VH-VL x I2C VH-VL in cynomolgus monkeys at dose levels of 60, 240 and 1000 ⁇ g/m 2 /24h is shown in Fig 32. These animals showed no signs at all of any T cell redistribution during the starting phase of treatment, i.e. T cell counts rather increased than decreased upon treatment initiation. Given that T cell redistribution is consistently observed in 100% of all patients without circulating target cells, upon treatment initiation with the conventional CD3 binding molecule (e.g.
- CD19xCD3 construct as described in WO 99/54440
- a context dependent CD3 epitope it was demonstrated that substantially less T cell redistribution in the absence of circulating target cells upon treatment initiation can be observed with a CD3 binding molecule of the invention directed and generated against an epitope of human an non- chimpanzee primate CD3 epsilon chain as defined by the amino acid sequence of anyone of SEQ ID NOs: 2, 4, 6, or 8 or a fragment thereof.
- CD3-binding molecules directed against a context-dependent CD3 epitope like the constructs described in WO 99/54440
- the binding molecules against context- independent CD3 epitopes as (inter alia) provided in any one of SEQ ID NOs: 2, 4, 6, or 8 (or fragments of these sequences) provide for this substantially less (detrimental and non-desired) T cell redistribution.
- T cell redistribution during the starting phase of treatment with CD3 binding molecules is a major risk factor for CNS adverse events
- the CD3 binding molecules provided herein and capable of recognizing a context independent CD3 epitope have a substantial advantage over the CD3 binding molecules known in the art and directed against context-dependent CD3 epitopes. Indeed none of the cynomolgus monkeys treated with MCSP-G4 VH-VL x I2C VH-VL showed any signs of CNS symptoms.
- the context-independence of the CD3 epitope is provided in this invention and corresponds to the first 27 N-terminal amino acids of CD3 epsilon) or fragments of this 27 amino acid stretch.
- This context-independent epitope is taken out of its native environment within the CD3 complex and fused to heterologous amino acid sequences without loss of its structural integrity.
- Anti-CD3 binding molecules as provided herein and generated (and directed) against a context-independent CD3 epitope provide for a surprising clinical improvement with regard to T cell redistribution and, thus, a more favorable safety profile.
- the CD3 binding molecules provided herein induce less allosteric changes in CD3 conformation than the conventional CD3 binding molecules (like molecules provided in WO 99/54440), which recognize context-dependent CD3 epitopes like molecules provided in WO 99/54440.
- the induction of intracellular NcK2 recruitment by the CD3 binding molecules provided herein is also reduced resulting in less isoform switch of T cell integrins and less adhesion of T cells to endothelial cells.
- preparations of CD3 binding molecules of the invention essentially consists of monomeric molecules. These monomeric molecules are even more efficient (than dimeric or multimeric molecules) in avoiding T cell redistribution and thus the risk of CNS adverse events during the starting phase of treatment.
- the coding sequence of human CD33 as published in GenBank was obtained by gene synthesis according to standard protocols.
- the gene synthesis fragment was designed as to contain first a Kozak site for eukaryotic expression of the construct, followed by a 19 amino acid immunoglobulin leader peptide, followed in frame by the coding sequence of the mature human CD33 protein, followed in frame by the coding sequence of serine glycine dipeptide, a5 histidine 6 -tag and a stop codon (the cDNA and amino acid sequence of the construct is listed under SEQ ID Nos 525 and 526).
- the gene synthesis fragment was also designed as to introduce restriction sites at the beginning and at the end of the fragment.
- A5 clone with sequence-verified nucleotide sequence was transfected into DHFR deficient CHO cells for eukaryotic expression of the construct.
- Eukaryotic protein expression in DHFR deficient CHO cells was performed as described by Kaufmann RJ. (1990) Methods Enzymol. 185, 537-566. Gene amplification of the construct was induced by increasing concentrations of methothrexate (MTX) to a final concentration0 of up to 20 nM MTX. 23.2. Generation of CHO cells expressing the extracellular domain of macaque CD33
- the cDNA sequence of macaque CD33 was obtained by a set of 3 PCRs on cDNA from macaque monkey bone marrow prepared according to standard protocols. The following reaction conditions: 1 cycle at 94°C for 3 minutes followed by 35 cycles with 94°C for 1 minute, 53°C for 1 minute and 72°C for 2 minutes followed by a terminal cycle of 72 0 C for 3 minutes and the following primers were used:
- forward primer 5'-gaggaattcaccatgccgctgctactgctgcccctgctgtgggcaggggccctggctatgg-3' reverse primer: 5'-gatttgtaactgtatttggtacttcc-3'
- forward primer 5'-gcaccaacctgacctgtcagg-3' reverse primer: 5'-agtgggtcgactcactgggtcctgacctctgagtattcg-3'
- reverse primer 5'-agtgggtcgactcactgggtcctgacctctgagtattcg-3'
- a cDNA fragment was obtained by gene synthesis according to standard protocols (the cDNA and amino acid sequence of the construct is listed under SEQ ID Nos 527 and 528).
- the coding sequence of macaque CD33 from amino acid +3 to +340 of the mature CD33 protein was fused into the coding sequence of human CD33 replacing the human coding sequence of the amino acids +3 to +340.
- the gene synthesis fragment was also designed as to contain a Kozak site for eukaryotic expression of the construct and restriction sites at the beginning and the end of the fragment containing the cDNA coding for essentially the whole extracellular domain of macaque CD33, the macaque CD33 transmembrane domain and a macaque-human chimeric intracellular CD33 domain.
- the introduced restriction sites Xbal at the 5' end and Sail at the 3' end, were utilised in the following cloning procedures.
- the gene synthesis fragment was then cloned via Xbal and Sail into a plasmid designated pEF-DHFR (pEF-DHFR is described in Kunststoff et al. Cancer Immunol lmmunother 50 (2001) 141-150). A sequence verified clone of this plasmid was used to transfect CHO/dhfr- cells as described above.
- bispecific antibody molecules each comprising a domain with a binding specificity cross-species specific for human and non-chimpanzee primate CD3 epsilon as well as a domain with a binding specificity cross-species specific for human and non-chimpanzee primate CD33, were designed as set out in the following Table 6:
- Table 6 Formats of anti-CD3 and anti-CD33 cross-species specific bispecific molecules
- variable light-chain (L) and variable heavy-chain (H) domains cross-species specific for human and macaque CD33 and the CD3 specific VH and VL combinations cross-species specific for human and macaque CD3 were obtained by gene synthesis.
- the gene synthesis fragments were designed as to contain first a Kozak site for eukaryotic expression of the construct, followed by a 19 amino acid immunoglobulin leader peptide, followed in frame by the coding sequence of the respective bispecific antibody molecule, followed in frame by the coding sequence of a histidine ⁇ -tag and a stop codon.
- the gene synthesis fragment was also designed as to introduce suitable restriction sites at the beginning and at the end of the fragment.
- the introduced restriction sites were utilized in the following cloning procedures.
- the gene synthesis fragment was cloned via these restriction sites into a plasmid designated pEF-DHFR (pEF-DHFR is described in Griffin et al. Cancer Immunol lmmunother 50 (2001) 141-150) following standard protocols.
- the aforementioned procedures were carried out according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York (2001)).
- a clone with sequence-verified nucleotide sequence was transfected into DHFR deficient CHO cells for eukaryotic expression of the construct.
- Eukaryotic protein expression in DHFR deficient CHO cells was performed as described by Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566. Gene amplification of the construct was induced by increasing concentrations of methothrexate (MTX) to a final concentration of up to 20 nM MTX.
- MTX methothrexate
- the bispecific antibody molecules are expressed in Chinese hamster ovary cells (CHO). Eukaryotic protein expression in DHFR deficient CHO cells was performed as described by Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566. Gene amplification of the constructs was induced by addition of increasing concentrations of MTX up to final concentrations of 20 nM MTX. After two passages of stationary culture the cells were grown in roller bottles with nucleoside-free HyQ PF CHO liquid soy medium (with 4.0 mM L-Glutamine with 0.1% Pluronic F - 68; HyClone) for 7 days before harvest. The cells were removed by centrifugation and the supernatant containing the expressed protein was stored at -20 0 C.
- CHO Chinese hamster ovary cells
- constructs were transiently expressed in HEK 293 cells. Transfection was performed with 293fectin reagent (Invitrogen, #12347-019) according to the manufacturer's protocol. Akta® Explorer System (GE Health Systems) and Unicorn® Software were used for chromatography. Immobilized metal affinity chromatography ("IMAC") was performed using a Fractogel EMD chelate® (Merck) which was loaded with ZnCI 2 according to the protocol provided by the manufacturer. The column was equilibrated with buffer A (2O mM sodium phosphate buffer pH 7.2, 0.1 M NaCI) and the cell culture supernatant (500 ml) was applied to the column (10 ml) at a flow rate of 3 ml/min.
- buffer A 2O mM sodium phosphate buffer pH 7.2, 0.1 M NaCI
- Eluted protein fractions from step 2 were pooled for further purification. All chemicals were of research grade and purchased from Sigma (Deisenhofen) or Merck (Darmstadt).
- the bispecific antibody protein was analyzed in SDS PAGE under reducing conditions performed with pre-cast 4-12% Bis Tris gels (Invitrogen). Sample preparation and application were performed according to the protocol provided by the manufacturer. The molecular weight was determined with MultiMark protein standard (Invitrogen). The gel was stained with colloidal Coomassie (Invitrogen protocol). The purity of the isolated protein is >95% as determined by SDS-PAGE.
- the bispecific antibody has a molecular weight of about 52 kDa under native conditions as determined by gel filtration in PBS. All constructs were purified according to this method.
- a FACS analysis was performed.
- CHO cells transfected with human CD33 as described in Example 23.1 and the human CD3 positive T cell leukemia cell line HPB-ALL (DSMZ, Braunschweig, ACC483) were used to test the binding to human antigens.
- the binding reactivity to macaque antigens was tested by using the generated macaque CD33 transfectant described in Example 23.2 and macaque PBMC (preparation of macaque PBMC was performed by Ficoll gradient centrifugation of peripheral blood from macaque monkeys according to standard protocolls).
- Bioactivity of the generated bispecific antibodies was analyzed by chromium 51 ( 51 Cr) release in vitro cytotoxicity assays using the CD33 positive cell lines described in Examples 23.1 and 23.2. As effector cells stimulated human CD4/CD56 depleted PBMC or the macaque T cell line 4119LnPx were used as specified in the respective figures.
- PBMC peripheral blood (30 - 50 ml human blood) by Ficoll gradient centrifugation according to standard protocols.
- a commercially available anti- CD3 specific antibody e.g. OKT3, Othoclone
- Unbound protein was removed by one washing step with PBS.
- the fresh PBMC were isolated from peripheral blood (30 - 50 ml human blood) by Ficoll gradient centrifugation according to standard protocols.
- the labeled target cells were washed 3 times with 5 ml RPMI and then used in the cytotoxicity assay.
- the assay was performed in a 96 well plate in a total volume of 250 ⁇ l supplemented RPMI (as above) with an E:T ratio of 10:1. 1 ⁇ g/ml of the cross- species specific bispecific antibody molecules and 20 threefold dilutions thereof were applied.
- the assay time was 18 hours and cytotoxicity was measured as relative values of released chromium in the supernatant related to the difference of maximum lysis (addition of Triton-X) and spontaneous lysis (without effector cells). All measurements were done in quadruplicates.
- pEF-DHFR pEF- DHFR is described in Kunststoff et al. Cancer Immunol lmmunother 50 (2001) 141-150) following standard protocols. The aforementioned procedures were carried out according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York (2001)).
- a clone with sequence-verified nucleotide sequence was transfected into DHFR deficient CHO cells for eukaryotic expression of the construct. Eukaryotic protein expression in DHFR deficient CHO cells was performed as described by Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566.
- Gene amplification of the construct was induced by increasing concentrations of methotrexate (MTX) to a final concentration of up to 20 nM MTX.
- MTX methotrexate
- the coding sequence of macaque CAIX as published in GenBank was obtained by gene synthesis according to standard protocols.
- the gene synthesis fragment was designed as to contain the coding sequence of the macaque CAIX protein including its leader peptide (the cDNA and amino acid sequence of the construct is listed under SEQ ID Nos 606 and 607).
- the gene synthesis fragment was also designed as to introduce restriction sites at the beginning and at the end of the fragment. The introduced restriction sites, Xbal at the 5' end and Sail at the 3' end, were utilized in the following cloning procedures.
- pEF-DHFR plasmid designated pEF-DHFR
- pEF-DHFR is described in Kunststoff et al. Cancer Immunol lmmunother 50 (2001) 141-150) following standard protocols. The afore-mentioned procedures were carried out according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York (2001)).
- a clone with sequence-verified nucleotide sequence was transfected into DHFR deficient CHO cells for eukaryotic expression of the construct. Eukaryotic protein expression in DHFR deficient CHO cells was performed as described by Kaufmann R.J.
- bispecific single chain antibody molecules each comprising a domain with a binding specificity cross-species specific for human and non-chimpanzee primate CD3epsilon as well as a domain with a binding specificity cross-species specific for human and non-chimpanzee primate CAIX, were designed as set out in the following Table 7:
- Table 7 Formats of anti-CD3 and anti-CAIX cross-species specific bispecific single chain antibody molecules
- variable light-chain (L) and variable heavy-chain (H) domains cross-species specific for human and macaque CAIX and the CD3 specific VH and VL combinations cross-species specific for human and macaque CD3 were obtained by gene synthesis.
- the gene synthesis fragments were designed as to contain first a Kozak site for eukaryotic expression of the construct, followed by a 19 amino acid immunoglobulin leader peptide, followed in frame by the coding sequence of the respective bispecific single chain antibody molecule, followed in frame by the coding sequence of a 6 histidine tag and a stop codon.
- the gene synthesis fragment was also designed as to introduce suitable restriction sites at the beginning and at the end of the fragment.
- the introduced restriction sites were utilized in the following cloning procedures.
- the gene synthesis fragment was cloned via these restriction sites into a plasmid designated pEF-DHFR (pEF-DHFR is described in Griffin et al. Cancer Immunol lmmunother 50 (2001) 141-150) following standard protocols.
- the aforementioned procedures were carried out according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York (2001)).
- a clone with sequence-verified nucleotide sequence was transfected into DHFR deficient CHO cells for eukaryotic expression of the construct.
- Eukaryotic protein expression in DHFR deficient CHO cells was performed as described by Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566. Gene amplification of the construct was induced by increasing concentrations of methothrexate (MTX) to a final concentration of up to 20 n M MTX.
- MTX methothrexate
- the bispecific single chain antibody molecules were expressed in Chinese hamster ovary cells (CHO). Eukaryotic protein expression in DHFR deficient CHO cells was performed as described by Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566. Gene amplification of the constructs was induced by addition of increasing concentrations of MTX up to final concentrations of 20 nM MTX. After two passages of stationary culture the cells were grown in roller bottles with nucleoside-free HyQ PF CHO liquid soy medium (with 4.0 mM L-Glutamine with 0.1% Pluronic F - 68; HyClone) for 7 days before harvest.
- CHO Chinese hamster ovary cells
- constructs were transiently expressed in HEK 293 cells. Transfection was performed with 293fectin reagent (Invitrogen, #12347-019) according to the manufacturer's protocol.
- IMAC Immobilized metal affinity chromatography
- Bound protein was eluted using a two step gradient of buffer B (20 mM sodium phosphate buffer pH 7.2, 0.1 M NaCI, 0.5 M Imidazole) according to the following: Step 1 : 20% buffer B in 6 column volumes Step 2: 100% buffer B in 6 column volumes Eluted protein fractions from step 2 were pooled for further purification. All chemicals are of research grade and purchased from Sigma (Deisenhofen) or Merck (Darmstadt).
- the bispecific single chain antibody has a molecular weight of about 52 kDa under native conditions as determined by gel filtration in PBS. All constructs were purified according to this method. Western Blot was performed using an Optitran® BA-S83 membrane and the Invitrogen Blot Module according to the protocol provided by the manufacturer. For detection of the bispecific single chain antibody protein antibodies an anti-His Tag antibody was used (Penta His, Qiagen). A Goat-anti-mouse Ig antibody labeled with alkaline phosphatase (AP) (Sigma) was used as secondary antibody and BCIP/NBT (Sigma) as substrate. A single band was detected at 52 kD corresponding to the purified bispecific single chain antibody. 24.5 Flow cytometric binding analysis of the CAIX and CD3 cross-species specific bispecific antibodies
- CHO cells transfected with human CAIX as described in Example 24.1 and the human CD3 positive T cell leukemia cell line HPB-ALL (DSMZ, Braunschweig, ACC483) were used to test the binding to human antigens.
- the binding reactivity to macaque antigens was tested by using the generated macaque CAIX transfectant described in Example 24.2 and a macaque T cell line 4119LnPx (kindly provided by Prof.
- Bioactivity of the generated bispecific single chain antibodies was analyzed by chromium 51 ( 51 Cr) release in vitro cytotoxicity assays using the CAIX positive cell lines described in Examples 24.1 and 24.2. As effector cells stimulated human CD4/CD56 depleted PBMC, stimulated human PBMC or the macaque T cell line 4119LnPx were used as specified in the respective figures.
- PBMC peripheral blood (30 - 50 ml human blood) by Ficoll gradient centrifugation according to standard protocols. 3 - 5 x 10 7 PBMC were added to the precoated petri dish in 120 ml of RPMI 1640 with stabilized glutamine / 10% FCS / IL-2 20 U/ml (Proleukin, Chiron) and stimulated for 2 days. On the third day the cells were collected and washed once with RPMI 1640. IL-2 was added to a final concentration of 20 U/ml and the cells were cultivated again for one day in the same cell culture medium as above.
- a commercially available anti-CD3 specific antibody e.g. OKT3, Othoclone
- 3 - 5 x 10 7 PBMC were added to the precoated petri dish in 120 ml of RPMI 1640 with stabilized glutamine / 10% FCS / IL-2 20 U/ml (Proleukin, Chiron) and stimulated for 2 days. On the third day the cells were collected and washed once with RPMI 1640. IL-2 was added to a final concentration of 20 U/ml and the cells were cultivated again for one day in the same cell culture medium as above.
- CTLs cytotoxic T lymphocytes
- Target cells were washed twice with PBS and labeled with 11.1 MBq 51 Cr in a final volume of 100 ⁇ l RPMI with 50% FCS for 45 minutes at 37 0 C. Subsequently the labeled target cells were washed 3 times with 5 ml RPMI and then used in the cytotoxicity assay.
- the assay was performed in a 96 well plate in a total volume of 250 ⁇ l supplemented RPMI (as above) with differing E:T ratios from 5:1 to 50:1 which are specified in the respective figures. 1 ⁇ g/ml of the cross-species specific bispecific single chain antibody molecules and 20 threefold dilutions thereof were applied.
- the assay time was 16 hours and cytotoxicity was measured as relative values of released chromium in the supernatant related to the difference of maximum lysis (addition of Triton-X) and spontaneous lysis (without effector cells). All measurements were done in quadruplicates. Measurement of chromium activity in the supernatants was performed with a Wizard 3" gamma counter (Perkin Elmer Life Sciences GmbH, KoIn, Germany). Analysis of the experimental data was performed with Prism 4 for Windows (version 4.02, GraphPad Software Inc., San Diego, California, USA). Sigmoidal dose response curves typically have R 2 values >0.90 as determined by the software. EC 50 values calculated by the analysis program were used for comparison of bioactivity.
- EpCAM-specific bispecific antibodies The basis for the construction of EpCAM-specific bispecific antibodies was the fully human, EpCAM specific antibody HD69 (Raum et al. Cancer Immunol lmmunother (2001) 50: 141 ff).
- the VH and the VL were PCR amplified from HD69 DNA according to standard methods.
- VH and VL were cloned subsequently into a plasmid, thus forming a functional scFv (orientation VH-Linker-VL) according to standard protocols.
- This scFv construct was then PCR amplified using primers introducing a N-terminal BsrGI and a C-terminal BspE1 restriction site useful for functional cloning into the bispecific antibody format
- the PCR product was then cloned into three suitable expression vectors, each comprising one CD3 specific VH and VL combination cross-species specific for human and macaque CD3 (H2C HL, F12Q HL and I2C, respectively), thus coding for three functional bispecific constructs HD69 HL x H2C HL, HD69 HL x F12Q HL and HD69 HL x I2C HL.
- the constructs were then transfected into DHFR-deficient CHO-cells by electroporation for stable transfection according to standard procedures, resulting in three transfected cell pools, each pool expressing one of the three bispecific products.
- Table 8 Formats of anti-EpCAM bispecific single chain antibody molecules comprising a human-cvnomolgus cross-specific anti-CD3 portion
- Bioactivity of the generated bispecific single chain antibodies was analyzed by chromium 51 release in vitro cytotoxicity assays using the transfected EpCAM- positive cell line CHO-EpCAM (expressing surface bound human EpCAM, published in Griffin et al. Cancer Immunol lmmunother (2001) 50: 141 ff) as target cells. As effector cells stimulated human CD8 positive T cells were used. As shown in Figure 37, all of the generated bispecific single chain antibody constructs revealed cytotoxic activity against human EpCAM-positive target cells elicited by human CD8+ cells. As a negative control, culture supernatant of untransfected CHO cells was used, which revealed no detectable cytotoxicity.
- CHO cells transfected with human EpCAM and human CD3 positive T cells in isolated human PBMCs were used to test the binding to human antigens.
- the binding reactivity to macaque CD3 antigen was tested by using a macaque T cell line 4119LnPx (kindly provided by Prof. Fickenscher, Hygiene Institute, Virology, Er Weg-Nuernberg; published in Knappe A, et al., and Fickenscher H., Blood 2000, 95, 3256-61). 200.000 cells of the respective cell lines were incubated for 30 min on ice with cell culture supernatant of transfected cells expressing the CD3 cross- species specific bispecific antibody constructs.
- the cells were washed twice in PBS with 2% FCS and binding of the construct was detected with a murine anti-His antibody (Penta His antibody; Qiagen; diluted 1 :20 in 50 ⁇ l PBS with 2% FCS). After washing, bound anti-His antibodies were detected with an Fc gamma-specific antibody (Dianova) conjugated to phycoerythrin, diluted 1 :100 in PBS with 2% FCS. Supernatant of untransfected CHO cells was used as negative control for binding to the T cell lines. Flow cytometry was performed on a FACS-Calibur apparatus and CellQuest software used to acquire and analyze the data (Becton Dickinson biosciences, Heidelberg).
- the gene synthesis fragment was designed as to contain first a Kozak site to allow for eukaryotic expression of the construct followed by the coding sequence of an 24 amino acid leader peptide followed in frame by the coding sequence of the human C-terminal extracellular domain, the transmembrane and cytoplasmic domain and a stop codon.
- 15 synthesis fragment was also designed as to introduce restriction sites at the beginning and at the end of the DNA fragment.
- a Xbal and Sail restiction enzyme recognition sites were added at the 5' and 3' end, respectively.
- the synthetic gene DNA was subsequently digested with Xbal and Sail, ligated into the appropriately digested expression vector pEF-DHFR, and transformed into E.coli.
- a 0 clone with verified nucleotide sequence (cDNA sequence and amino acid sequence of the recombinant construct is listed under SEQ ID NOs 599 and 598) was transfected into DHFR deficient CHO cells for eukaryotic expression of the construct. Eukaryotic protein expression in DHFR deficient CHO cells was performed as described above. 5 26.2 Generation and expression of the of cynomolgus EGFR vlll
- the cynomolgus EGFR vlll variant was created by deletion of the 267 amino acids of the extracellular domain and insertion of the new amino acid glycine at the fusion site (amino acid no. 6).
- the gene synthesis fragment was designed as to contain first a Kozak site to allow for eukaryotic expression of the 0 construct followed by the coding sequence of an 24 amino acid leader peptide followed in frame by the coding sequence of the cynomolgus C-terminal extracellular domain, the transmembrane and cytoplasmic domain (both human) and a stop codon.
- the gene synthesis fragment was also designed as to introduce restriction sites at the beginning and at the end of the DNA fragment.
- the fragment was digested with Xbal / Sail and cloned into pEF-DHFR following standard protocols.
- a sequence verified plasmid was used to transfect CHO/dhfr- cells (ATCC No.
- CRL 9096 cultivated in RPMI 1640 with stabilized glutamine obtained from Biochrom AG Berlin, Germany, supplemented with 10% FCS, 1% penicillin/streptomycin all obtained from Biochrom AG Berlin, Germany and nucleosides from a stock solution of cell culture grade reagents obtained from Sigma- Aldrich Chemie GmbH, Taufkirchen, Germany, to a final concentration of 10 ⁇ g/ml Adenosine, 10 ⁇ g/ml Deoxyadenosine and 10 ⁇ g/ml Thymidine, in an incubator at 37 0 C, 95% humidity and 7% CO2).
- Transfection was performed using the PolyFect Transfection Reagent (Qiagen GmbH, Hilden, Germany) and 5 ⁇ g of plasmid DNA according to the manufacturer's protocol. After a cultivation of 24 hours, cells were washed once with PBS and again cultivated in the aforementioned cell culture medium except that the medium was not supplemented with nucleosides and dialyzed FCS (obtained from Biochrom AG Berlin, Germany) was used. Thus the cell culture medium did not contain nucleosides and thereby selection was applied on the transfected cells. Approximately 14 days after transfection the outgrowth of resistant cells was observed. After an additional 7 to 14 days the transfectants were tested positive for expression of the construct via FACS.
- bispecific single chain antibody molecules each comprising a domain with a binding specificity for the human and the non-chimpanzee primate CD3 antigen as well as a domain with a binding specificity for the human and the non-chimpanzee primate EGFRvIII antigen, were designed as set out in the following Table 9:
- Table 9 Formats of anti-CD3 and anti-EGFRvlll cross-species specific bispecific single chain antibody molecules SEQ ID Formats of protein constructs (nucl/prot) (N * C)
- variable light-chain (L) and variable heavy-chain (H) domains cross-species specific for human and cynomolgus EGFR vlll and the CD3 specific VH and VL combinations cross-species specific for human and macaque CD3 were obtained by gene synthesis.
- the gene synthesis fragments were designed as to contain first a Kozak site for eukaryotic expression of the construct, followed by a 19 amino acid immunoglobulin leader peptide, followed in frame by the coding sequence of the respective bispecific single chain antibody molecule, followed in frame by the coding sequence of a 6 histidine tag and a stop codon.
- the gene synthesis fragment was also designed as to introduce suitable restriction sites at the beginning and at the end of the fragment.
- the introduced restriction sites were utilized in the following cloning procedures.
- the gene synthesis fragment was cloned via these restriction sites into a plasmid designated pEF-DHFR following standard protocols.
- a clone with sequence-verified nucleotide sequence was transfected into DHFR deficient CHO cells for eukaryotic expression of the construct.
- Eukaryotic protein expression in DHFR deficient CHO cells was performed as described by Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566.
- Gene amplification of the construct was induced by increasing concentrations of methothrexate (MTX) to a final concentration of up to 20 nM MTX.
- methothrexate MTX
- the constructs were transfected into DHFR-deficient CHO-cells in a transient manner according to standard protocols.
- the FACS binding experiments were performed with the human EGFRvIII transfected CHO cell line to assess the binding capability to the human EGFRvIII antigen.
- the cross-species specificity to cynomolgus tissue was tested by deploying the CHO cells transfected with the cynomolgus EGFRvIII.
- the same changes in cell lines apply to the cytotoxicity assays performed with the EGFRvIII and CD3 cross- species specific bispecific single chain antibodies. Apart from this the assays were performed as described in examples 4 and 5.
- the generated EGFRvIII and CD3 cross-species specific bispecific single chain antibodies demonstrated binding to both the human and cynomolgus antigens and proved to be fully cross-species specific.
- all of the generated cross-species specific bispecific single chain antibody constructs revealed cytotoxic activity against human EGFRvIII positive target cells elicited by human CD8+ cells and cynomolgus EGFRvIII positive target cells elicited by the macaque T cell line 4119LnPx. As a negative control, an irrelevant bispecific single chain antibody has been used.
- mice cell line J558L obtained from lnterlab Project, lstituto Nazionale per Ia Ricerca sul Cancro, Genova, Italy, ECACC 88032902
- a spontaneous heavy chain- loss-variant myeloma cell line that synthesizes and secretes a lambda light chain was used to be complemented by a membrane bound heavy chain variant of the human and macaque IgE, respectively.
- synthetic molecules were obtained by gene synthesis according to standard protocols (the nucleotide sequences of the constructs are listed under SEQ ID Nos 595 and 594).
- the coding sequence for human and macaque CD3 epsilon chain was fused to the human transmembrane region of IgE, respectively.
- the built in specificity of the VH chain is directed against the hapten (4-hydroxy-3- nitro-phenyl)acetyl) (NP).
- the gene synthesis fragment was also designed as to contain a Kozak site for eukaryotic expression of the construct and a immunoglobulin leader and restriction sites at the beginning and the end of the DNA.
- the introduced restriction sites EcoRI at the 5' end and Sail at the 3' end were utilized during the cloning step into the expression plasmid designated pEF-DHFR.
- bispecific single chain antibody molecules each comprising a domain with a binding specificity for the human and the non-chimpanzee primate CD3 antigen as well as a domain with a binding specificity for the human and non-chimpanzee primate IgE antigen, were designed as set out in the following Table 10:
- Table 10 Formats of anti-CD3 and anti-lgE cross-species specific bispecific single chain antibody molecules SEQ ID Formats of protein constructs (nucl/prot) (N -» C)
- variable light-chain (L) and variable heavy-chain (H) domains cross-species specific for human and macaque IgE and the CD3 specific VH and VL combinations cross-species specific for human and macaque CD3 were obtained by gene synthesis.
- the gene synthesis fragments were designed as to contain first a Kozak site for eukaryotic expression of the construct, followed by a 19 amino acid immunoglobulin leader peptide, followed in frame by the coding sequence of the respective bispecific single chain antibody molecule, followed in frame by the coding sequence of a 6 histidine tag and a stop codon.
- the gene synthesis fragment was also designed as to introduce suitable restriction sites at the beginning and at the end of the fragment.
- the introduced restriction sites were utilized in the following cloning procedures.
- the gene synthesis fragment was cloned via these restriction sites into a plasmid designated pEF-DHFR following standard protocols.
- a clone with sequence-verified nucleotide sequence was transfected into DHFR deficient CHO cells for eukaryotic expression of the construct. Eukaryotic protein expression in DHFR deficient CHO cells was performed as described by Kaufmann RJ. (1990) Methods Enzymol. 185, 537-566.
- Gene amplification of the construct was induced by increasing concentrations of methothrexate (MTX) to a final concentration of up to 20 nM MTX.
- MTX methothrexate
- the constructs were transfected into DHFR-deficient CHO-cells in a transient manner according to standard protocols.
- the FACS binding experiments were performed with the human IgE transfected J558L cell line to assess the binding capability to the human IgE .
- the cross-species specificity to macaque IgE positive cells was tested by deploying the J558L cells transfected with the macaque IgE.
- the same changes in cell lines apply to the The J558L cell line.
- the binding experiments as well as the cytotoxicity assays were performed as described above.
- the generated IgE and CD3 cross-species specific bispecific single chain antibodies demonstrated binding to both the human and cynomolgus antigens and proved to be fully cross-species specific.
- the coding sequence of human CD44 as published in GenBank (Accession number AJ251595) and also containing the CD44v6 exon was obtained by gene synthesis according to standard protocols.
- the gene synthesis fragment was designed as to contain first a Kozak site for eukaryotic expression of the construct, followed by the coding sequence of the human CD44 protein and a stop codon (the cDNA and amino acid sequence of the construct is listed under SEQ ID Nos 608 and 609).
- the gene synthesis fragment was also designed as to introduce restriction sites at the beginning and at the end of the fragment. The introduced restriction sites, EcoRI at the 5' end and Sail at the 3' end, were utilized in the following cloning procedures.
- pEF-DHFR was described in Kunststoff et al. Cancer Immunol lmmunother 50 (2001) 141-150) following standard protocols. The afore-mentioned procedures were carried out according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York (2001)).
- a clone with sequence-verified nucleotide sequence was transfected into DHFR deficient CHO cells for eukaryotic expression of the construct. Eukaryotic protein expression in DHFR deficient CHO cells was performed as described by Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566.
- Gene amplification of the construct was induced by increasing concentrations of methotrexate (MTX) to a final concentration of up to 20 nM MTX.
- MTX methotrexate
- the coding sequence of human CD44 as published in GenBank (Accession number AJ251595) deleted for the sequence coding the v6 exon was obtained by gene synthesis according to standard protocols.
- the gene synthesis fragment was designed as to contain first a Kozak site for eukaryotic expression of the construct, followed by the coding sequence of the human CD44 protein without the v6 exon and a stop codon (the cDNA and amino acid sequence of the construct is listed under SEQ ID Nos 610 and 611).
- the gene synthesis fragment was also designed as to introduce restriction sites at the beginning and at the end of the fragment. The introduced restriction sites, EcoRI at the 5' end and Sail at the 3' end, were utilized in the following cloning procedures.
- pEF-DHFR The gene synthesis fragment was cloned via EcoRI and Sail into a plasmid designated pEF-DHFR (pEF-DHFR is described in Griffin et al. Cancer Immunol lmmunother 50 (2001) 141-150) following standard protocols. The afore-mentioned procedures were carried out according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York (2001)). A clone with sequence-verified nucleotide sequence was transfected into DHFR deficient CHO cells for eukaryotic expression of the construct. Eukaryotic protein expression in DHFR deficient CHO cells was performed as described by Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566. Gene amplification of the construct was induced by increasing concentrations of methotrexate (MTX) to a final concentration of up to 20 nM MTX.
- the gene synthesis fragment was also designed as to contain a Kozak site for eukaryotic expression of the construct and restriction sites at the beginning and the end of the fragment.
- the gene synthesis fragment was then cloned via EcoRI and Sail into a plasmid designated pEF-DHFR (pEF-DHFR is described in Jardin et al. Cancer Immunol lmmunother 50 (2001) 141-150).
- pEF-DHFR is described in Kunststoff et al. Cancer Immunol lmmunother 50 (2001) 141-150).
- a sequence verified clone of this plasmid was used to transfect CHO/dhfr- cells as described above. 28.4 Generation of CD44v6 and CD3 cross-species specific bispecific single chain molecules
- bispecific single chain antibody molecules each comprising a domain with a binding specificity cross-species specific for human and non-chimpanzee primate CD3epsilon as well as a domain with a binding specificity cross-species specific for human and non-chimpanzee primate CD44v6, are designed as set out in the following Table 11 :
- Table 11 Formats of anti-CD3 and anti-CD44v6 cross-species specific bispecific single chain antibody molecules
- variable light-chain (L) and variable heavy-chain (H) domains cross-species specific for human and macaque CD44 and the CD3 specific VH and VL combinations cross-species specific for human and macaque CD3 are obtained by gene synthesis.
- the gene synthesis fragments were designed as to contain first a Kozak site for eukaryotic expression of the construct, followed by a 19 amino acid immunoglobulin leader peptide, followed in frame by the coding sequence of the respective bispecific single chain antibody molecule, followed in frame by the coding sequence of a histidine ⁇ -tag and a stop codon.
- the gene synthesis fragment was also designed as to introduce suitable restriction sites at the beginning and at the end of the fragment.
- the introduced restriction sites were utilized in the following cloning procedures.
- the gene synthesis fragment was cloned via these restriction sites into a plasmid designated pEF-DHFR (pEF-DHFR is described in Griffin et al. Cancer Immunol lmmunother 50 (2001) 141-150) following standard protocols.
- the aforementioned procedures were carried out according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York (2001)).
- a clone with sequence-verified nucleotide sequence was transfected into DHFR deficient CHO cells for eukaryotic expression of the construct.
- Eukaryotic protein expression in DHFR deficient CHO cells was performed as described by Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566. Gene amplification of the construct was induced by increasing concentrations of methotrexate (MTX) to a final concentration of up to 20 n M MTX.
- MTX methotrexate
- the bispecific single chain antibody molecules were expressed in Chinese hamster ovary cells (CHO). Eukaryotic protein expression in DHFR deficient CHO cells was performed as described by Kaufmann RJ. (1990) Methods Enzymol. 185, 537-566. Gene amplification of the constructs was induced by addition of increasing concentrations of MTX up to final concentrations of 20 nM MTX. After two passages of stationary culture the cells were grown in roller bottles with nucleoside-free HyQ PF CHO liquid soy medium (with 4.0 mM L-Glutamine with 0.1% Pluronic F - 68; HyClone) for 7 days before harvest.
- CHO Chinese hamster ovary cells
- constructs were transiently expressed in HEK 293 cells. Transfection was performed with 293fectin reagent (Invitrogen, #12347-019) according to the manufacturer's protocol.
- IMAC Immobilized metal affinity chromatography
- Bound protein was eluted using a two step gradient of buffer B (20 mM sodium phosphate buffer pH 7.2, 0.1 M NaCI, 0.5 M Imidazole) according to the following: Step 1 : 20% buffer B in 6 column volumes Step 2: 100% buffer B in 6 column volumes Eluted protein fractions from step 2 were pooled for further purification. All chemicals were of research grade and purchased from Sigma (Deisenhofen) or Merck (Darmstadt).
- the bispecific single chain antibody has a molecular weight of about 52 kDa under native conditions as determined by gel filtration in PBS. All constructs were purified according to this method. Western Blot was performed using an Optitran® BA-S83 membrane and the Invitrogen Blot Module according to the protocol provided by the manufacturer. The antibodies used were directed against the histidine ⁇ -tag (Penta His, Qiagen) and Goat-anti-mouse Ig labeled with alkaline phosphatase (AP) (Sigma), and BCIP/NBT (Sigma) as substrate. A single band was detected at 52 kD corresponding to the purified bispecific single chain antibody.
- a FACS analysis was performed.
- CHO cells transfected with human CD44 as described in Example 28.1 and the human CD3 positive T cell leukemia cell line HPB-ALL (DSMZ, Braunschweig, ACC483) were used to test the binding to human antigens.
- the binding reactivity to macaque antigens was tested by using the generated macaque CD44 transfectant described in Example 28.3 and macaque PBMC (preparation of macaque PBMC was performed by Ficoll gradient centrifugation of peripheral blood from macaque monkeys according to standard protocols).
- v6 exon of CD44 was tested by using the transfected CHO cells expressing human CD44 without the v6 exon generated as described in Example 28.2. 200.000 cells of the respective cell lines or macaque PBMC were incubated for 30 min. on ice with 50 ⁇ l of the purified protein of the cross-species specific bispecific antibody constructs (5 ⁇ g/ml) or 50 ⁇ l of a commercially available anti-CD44 antibody (Becton Dickinson biosciences, Heidelberg; also 5 ⁇ g/ml).
- the cells were washed twice in PBS with 2% FCS and binding of the construct was detected with a murine anti-His antibody (Penta His antibody; Qiagen; diluted 1 :20 in 50 ⁇ l PBS with 2% FCS).
- the anti-His antibody was omitted for the samples incubated with the commercially available anti-CD44 antibody.
- bound anti-His antibody or anti-CD44 antibody was detected with an Fc gamma-specific antibody (Dianova) conjugated to phycoerythrin, diluted 1 :100 in PBS with 2% FCS.
- PBS with 2% FCS was used as negative control.
- Flow cytometry was performed on a FACS-Calibur apparatus; the CellQuest software was used to acquire and analyze the data (Becton Dickinson biosciences, Heidelberg). FACS staining and measuring of the fluorescence intensity were performed as described in Current Protocols in Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober, Wiley-lnterscience, 2002).
- Bioactivity of the generated bispecific single chain antibodies was analyzed by chromium 51 ( 51 Cr) release in vitro cytotoxicity assays using the CD44 positive cell line described in Example 28.1. As effector cells stimulated human CD4/CD56 depleted PBMC were used.
- PBMC peripheral blood (30 - 50 ml human blood) by Ficoll gradient centrifugation according to standard protocols. 3 - 5 x 10 7 PBMC were added to the precoated petri dish in 120 ml of RPMI 1640 with stabilized glutamine / 10% FCS / IL-2 20 U/ml (Proleukin, Chiron) and stimulated for 2 days. On the third day the cells were collected and washed once with RPMI 1640. IL-2 was added to a final concentration of 20 U/ml and the cells were cultivated again for one day in the same cell culture medium as above.
- a commercially available anti-CD3 specific antibody e.g. OKT3, Orthoclone
- CD8+ cytotoxic T lymphocytes were enriched.
- Target cells were washed twice with PBS and labeled with 11.1 MBq 51 Cr in a final volume of 100 ⁇ l RPMI with 50% FCS for 45 minutes at 37°C. Subsequently the labeled target cells were washed 3 times with 5 ml RPMI and then used in the cytotoxicity assay. The assay was performed in a 96 well plate in a total volume of 250 ⁇ l supplemented RPMI (as above) with an E:T ratio of 10:1.
- the gene synthesis fragment was cloned via EcoRI and Sail into a plasmid designated pEF-DHFR (pEF-DHFR is described in Kunststoff et al. Cancer Immunol lmmunother 50 (2001) 141-150) following standard protocols.
- forward primer 5'-gcttcttccattctgtctgcccagcagg-3' reverse primer: 5'-ggtaggggacacagcgggcacaggagttgg-3'
- forward primer 5'-cctggcatgatctgtgccacatcagcc-3' reverse primer: 5'-gcgttgagctcctcctgggtctgg-3'
- PCR reactions were performed under addition of PCR grade betain to a final concentration of 1 M. Those PCRs generate four overlapping fragments, which were isolated and sequenced according to standard protocols using the PCR primers, and thereby provide a portion of the cDNA sequence coding macaque CD30 from codon 12 of the leader peptide to codon 562 of the mature protein.
- a cDNA fragment was obtained by gene synthesis according to standard protocols (the cDNA and amino acid sequence of the construct is listed under SEQ ID Nos 1105 and 1106).
- the coding sequence for macaque CD30 from amino acid 12 of the leader peptide to amino acid 562 of the mature CD30 protein was fused into the coding sequence of human CD30 replacing the respective part of the human coding sequence.
- the gene synthesis fragment was also designed as to contain a Kozak site for eukaryotic expression of the construct and restriction sites at the beginning and the end of the fragment containing the cDNA coding for the whole extracellular domain of macaque CD30, the macaque CD30 transmembrane domain and a macaque-human chimeric intracellular CD30 domain.
- the introduced restriction sites Xbal at the 5' end and Sail at the 3' end, were utilized in the following cloning procedures.
- pEF-DHFR plasmid designated pEF-DHFR
- pEF-DHFR is described in Griffin et al. Cancer Immunol lmmunother 50 (2001) 141-150).
- a sequence verified clone of this plasmid was used to transfect CHO/dhfr- cells as described above.
- bispecific single chain antibody molecules each comprising a domain with a binding specificity cross-species specific for human and non-chimpanzee primate CD3epsilon as well as a domain with a binding specificity cross-species specific for human and non-chimpanzee primate CD30, were designed as set out in the following Table 12:
- Table 12 Formats of anti-CD3 and anti-CD30 cross-species specific bispecific single chain antibody molecules
- variable light-chain (L) and variable heavy-chain (H) domains cross-species specific for human and macaque CD30 and the CD3 specific VH and VL combinations cross-species specific for human and macaque CD3 were obtained by gene synthesis.
- the gene synthesis fragments were designed as to contain first a Kozak site for eukaryotic expression of the construct, followed by a 19 amino acid immunoglobulin leader peptide, followed in frame by the coding sequence of the respective bispecific single chain antibody molecule, followed in frame by the coding sequence of a 6 histidine tag and a stop codon.
- the gene synthesis fragment was also designed as to introduce suitable restriction sites at the beginning and at the end of the fragment.
- the introduced restriction sites were utilised in the following cloning procedures.
- the gene synthesis fragment was cloned via these restriction sites into a plasmid designated pEF-DHFR (pEF-DHFR is described in Griffin et al. Cancer Immunol lmmunother 50 (2001) 141-150) following standard protocols.
- the aforementioned procedures were carried out according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York (2001)).
- a clone with sequence-verified nucleotide sequence was transfected into DHFR deficient CHO cells for eukaryotic expression of the construct.
- Eukaryotic protein expression in DHFR deficient CHO cells was performed as described by Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566. Gene amplification of the construct was induced by increasing concentrations of methotrexate (MTX) to a final concentration of up to 20 nM MTX.
- MTX methotrexate
- the bispecific single chain antibody molecules were expressed in Chinese hamster ovary cells (CHO). Eukaryotic protein expression in DHFR deficient CHO cells was performed as described by Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566. Gene amplification of the constructs was induced by addition of increasing concentrations of MTX up to final concentrations of 20 nM MTX. After two passages of stationary culture the cells were grown in roller bottles with nucleoside-free HyQ PF CHO liquid soy medium (with 4.0 mM L-Glutamine with 0.1% Pluronic F - 68; HyClone) for 7 days before harvest.
- CHO Chinese hamster ovary cells
- constructs were transiently expressed in HEK 293 cells. Transfection was performed with 293fectin reagent (Invitrogen, #12347-019) according to the manufacturer's protocol.
- IMAC Immobilized metal affinity chromatography
- Bound protein was eluted using a two step gradient of buffer B (20 mM sodium phosphate buffer pH 7.2, 0.1 M NaCI, 0.5 M Imidazole) according to the following procedure: Step 1 : 20% buffer B in 6 column volumes Step 2: 100% buffer B in 6 column volumes Eluted protein fractions from step 2 were pooled for further purification. All chemicals are of research grade and purchased from Sigma (Deisenhofen) or Merck (Darmstadt).
- the bispecific single chain antibody has a molecular weight of about 52 kDa under native conditions as determined by gel filtration in PBS. All constructs were purified according to this method. Western Blot was performed using an Optitran® BA-S83 membrane and the Invitrogen Blot Module according to the protocol provided by the manufacturer. The antibodies used were directed against the His Tag (Penta His, Qiagen) and Goat- anti-mouse Ig labeled with alkaline phosphatase (AP) (Sigma), and BCIP/NBT (Sigma) as substrate. A single band was detected at 52 kD corresponding to the purified bispecific single chain antibody.
- the binding reactivity to macaque antigens was tested by using the generated macaque CD30 transfectant described in Example 29.2 and a macaque T cell line 4119LnPx (kindly provided by Prof Fickenscher, Hygiene Institute, Virology, Er Weg-Nuernberg; published in Knappe A, et al., and Fickenscher H., Blood 2000, 95, 3256-61). 200.000 cells of the respective cell lines were incubated for 30 min on ice with 50 ⁇ l of the purified protein of the cross-species specific bispecific antibody constructs (5 ⁇ g/ml).
- the cells were washed twice in PBS with 2% FCS and binding of the construct was detected with a murine Penta His antibody (Qiagen; diluted 1 :20 in 50 ⁇ l PBS with 2% FCS). After washing, bound anti His antibodies were detected with an Fc gamma-specific antibody (Dianova) conjugated to phycoerythrin, diluted 1 :100 in PBS with 2% FCS. PBS with 2% FCS was used as a negative control.
- a murine Penta His antibody Qiagen; diluted 1 :20 in 50 ⁇ l PBS with 2% FCS.
- Fc gamma-specific antibody Dianova conjugated to phycoerythrin
- Bioactivity of the generated bispecific single chain antibodies was analyzed by chromium 51 ( 51 Cr) release in vitro cytotoxicity assays using the human CD30 positive B cell line MEC-1 (DSMZ, Braunschweig, ACC 497) and the CHO cells transfected with macaque CD30 described in Example 29.2.
- Generation of stimulated human PBMC was performed as follows:
- a Petri dish (145 mm diameter, Greiner bio-one GmbH, Kremsm ⁇ nster) was coated with a commercially available anti-CD3 specific antibody (e.g. OKT3, Orthoclone) in a final concentration of 1 ⁇ g/ml for 1 hour at 37°C. Unbound protein was removed by one washing step with PBS.
- the fresh PBMC were isolated from peripheral blood (30 - 50 ml human blood) by Ficoll gradient centrifugation according to standard protocols. 3 - 5 x 10 7 PBMC were added to the precoated petri dish in 120 ml of RPMI 1640 with stabilized glutamine / 10% FCS / IL-2 20 U/ml (Proleukin, Chiron) and stimulated for 2 days. On the third day the cells were collected and washed once with RPMI 1640. IL-2 was added to a final concentration of 20 U/ml and the cells were cultivated again for one day in the same cell culture medium as above.
- CD8+ cytotoxic T lymphocytes were enriched.
- Target cells were washed twice with PBS and labelled with 11.1 MBq 51 Cr in a final volume of 100 ⁇ l RPMI with 50% FCS for 60 minutes at 37°C. Subsequently the labelled target cells were washed 3 times with 5 ml RPMI and then used in the cytotoxicity assay. The assay was performed in a 96 well plate in a total volume of 250 ⁇ l supplemented RPMI (as above) with an E:T ratio of 10:1. 1 ⁇ g/ml of the cross- species specific bispecific single chain antibody molecules and 20 threefold dilutions thereof were applied.
- the assay time was 4 hours for the assay using MEC-1 and human CD4/CD56 depleted PBMC and 18 hours for the assay using the macaque CD30 transfected CHO and the macaque T cell line 4119LnPx.
- Cytotoxicity was measured as relative values of released chromium in the supernatant related to the difference of maximum lysis (addition of Triton-X) and spontaneous lysis (without effector cells). All measurements were done in quadruplicates. Measurement of chromium activity in the supernatants was performed with a Wizard 3" gammacounter (Perkin Elmer Life Sciences GmbH, KoIn, Germany).
- the coding sequence of human HER2 as published in GenBank was obtained by gene synthesis according to standard protocols.
- the gene synthesis fragment was designed as to contain the coding sequence of the human HER2 protein including its leader peptide (the cDNA and amino acid sequence of the construct is listed under SEQ ID Nos 1107 and 1108.
- the gene synthesis fragment was also designed as to introduce restriction sites at the beginning and at the end of the fragment. The introduced restriction sites, Xbal at the 5' end and Sail at the 3' end, were utilised in the following cloning procedures.
- pEF-DHFR plasmid designated pEF-DHFR
- pEF-DHFR is described in Kunststoff et al. Cancer Immunol lmmunother 50 (2001) 141-150) following standard protocols. The aforementioned procedures were carried out according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York (2001)).
- a clone with sequence-verified nucleotide sequence was transfected into DHFR deficient CHO cells for eukaryotic expression of the construct. Eukaryotic protein expression in DHFR deficient CHO cells was performed as described by Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566.
- Gene amplification of the construct was induced by increasing concentrations of methotrexate (MTX) to a final concentration of up to 20 nM MTX.
- MTX methotrexate
- the coding sequence of human HER2 as described above was modified to encode the amino acids 123 to 1038 of the macaque HER2 protein as published in GenBank (Accession number XP_001090430).
- the coding sequence for this chimeric protein was obtained by gene synthesis according to standard protocols (the cDNA and amino acid sequence of the construct is listed under SEQ ID Nos 1109 and 1110).
- the gene synthesis fragment was also designed as to contain a Kozak site for eukaryotic expression of the construct and restriction sites at the beginning and the end of the fragment.
- the introduced restriction sites Xbal at the 5' end and Sail at the 3' end, were utilized in the following cloning procedures.
- pEF-DHFR plasmid designated pEF-DHFR
- pEF-DHFR is described in Griffin et al. Cancer Immunol lmmunother 50 (2001) 141- 150).
- a sequence verified clone of this plasmid was used to transfect CHO/dhfr- cells as described above.
- bispecific single chain antibody molecules each comprising a domain with a binding specificity cross-species specific for human and non-chimpanzee primate CD3epsilon as well as a domain with a binding specificity cross-species specific for human and macaque HER2, are designed as set out in the following Table 13:
- Table 13 Formats of anti-CD3 and anti-HER2 cross-species specific bispecific single chain antibody molecules SEQ ID Formats of protein constructs (nucl/prot) (N -* C)
- variable light-chain (L) and variable heavy-chain (H) domains cross-species specific for human and macaque HER2 and the CD3 specific VH and VL combinations cross-species specific for human and macaque CD3 were obtained by gene synthesis.
- the gene synthesis fragments were designed as to contain first a Kozak site for eukaryotic expression of the construct, followed by a 19 amino acid immunoglobulin leader peptide, followed in frame by the coding sequence of the respective bispecific single chain antibody molecule, followed in frame by the coding sequence of a 6 histidine tag and a stop codon.
- the gene synthesis fragment was also designed as to introduce suitable restriction sites at the beginning and at the end of the fragment.
- the introduced restriction sites were utilised in the following cloning procedures.
- the gene synthesis fragment was cloned via these restriction sites into a plasmid designated pEF-DHFR (pEF-DHFR is described in Griffin et al. Cancer Immunol lmmunother 50 (2001) 141-150) following standard protocols.
- the aforementioned procedures were carried out according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York (2001)).
- a clone with sequence-verified nucleotide sequence was transfected into DHFR deficient CHO cells for eukaryotic expression of the construct.
- Eukaryotic protein expression in DHFR deficient CHO cells was performed as described by Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566. Gene amplification of the construct was induced by increasing concentrations of methothrexate (MTX) to a final concentration of up to 20 nM MTX.
- MTX methothrexate
- the bispecific single chain antibody molecules were expressed in Chinese hamster ovary cells (CHO). Eukaryotic protein expression in DHFR deficient CHO cells was performed as described by Kaufmann R.J. (1990) Methods Enzymol. 185, 537-566. Gene amplification of the constructs was induced by addition of increasing concentrations of MTX up to final concentrations of 20 nM MTX. After two passages of stationary culture the cells are grown in roller bottles with nucleoside-free HyQ PF CHO liquid soy medium (with 4.0 mM L-Glutamine with 0.1 % Pluronic F - 68; HyClone) for 7 days before harvest.
- nucleoside-free HyQ PF CHO liquid soy medium with 4.0 mM L-Glutamine with 0.1 % Pluronic F - 68; HyClone
- the cells were removed by centrifugation and the supernatant containing the expressed protein was stored at -80 0 C.
- Transfection was performed with 293fectin reagent (Invitrogen, #12347-019) according to the manufacturer's protocol.
- Akta® Explorer System (GE Health Systems) and Unicorn® Software were used for chromatography.
- Immobilized metal affinity chromatography (“IMAC") was performed using a Fractogel EMD chelate® (Merck) which was loaded with ZnCI 2 according to the protocol provided by the manufacturer.
- the column was equilibrated with buffer A (2O mM sodium phosphate buffer pH 7.2, 0.1 M NaCI) and the cell culture supernatant (500 ml) was applied to the column (10 ml) at a flow rate of 3 ml/min. The column was washed with buffer A to remove unbound sample. Bound protein was eluted using a two step gradient of buffer B (20 mM sodium phosphate buffer pH 7.2, 0.1 M NaCI, 0.5 M Imidazole) according to the following: Step 1 : 20% buffer B in 6 column volumes Step 2: 100% buffer B in 6 column volumes
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ES08735001T ES2432792T5 (en) | 2007-04-03 | 2008-04-03 | Cross-species specific CD3-epsilon binding domain |
JP2010501434A JP2010524851A (en) | 2007-04-03 | 2008-04-03 | Species-specific binding domains |
KR1020177023629A KR101940944B1 (en) | 2007-04-03 | 2008-04-03 | Cross-species-specific cd3-epsilon binding domain |
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EP18184899.5A EP3461842A1 (en) | 2007-04-03 | 2008-04-03 | Cross-species-specific binding domain |
CA2683370A CA2683370C (en) | 2007-04-03 | 2008-04-03 | Cross-species-specific binding domain |
EP12163656.7A EP2520590B1 (en) | 2007-04-03 | 2008-04-03 | Cross-species-specific binding domain |
RS20130463A RS53008B2 (en) | 2007-04-03 | 2008-04-03 | Cross-species-specific cd3-epsilon binding domain |
NZ580755A NZ580755A (en) | 2007-04-03 | 2008-04-03 | Cross-species-specific cd3-epsilon binding domain |
RU2009136912/10A RU2561457C2 (en) | 2007-04-03 | 2008-04-03 | Cd3-epsilon-binding domain having interspecies specificity |
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EP08735001.3A EP2155783B2 (en) | 2007-04-03 | 2008-04-03 | Cross-species-specific cd3-epsilon binding domain |
BRPI0809594A BRPI0809594A2 (en) | 2007-04-03 | 2008-04-03 | polypeptide, nucleic acid sequence, vector, host, process for producing a polypeptide, pharmaceutical composition, use of a polypeptide, method for preventing, treating or ameliorating a disease in an individual in need thereof, kit, method for the identification of a polypeptide (s) |
US12/594,713 US20100150918A1 (en) | 2007-04-03 | 2008-04-03 | Cross-species-specific binding domain |
PL08735001.3T PL2155783T5 (en) | 2007-04-03 | 2008-04-03 | Cross-species-specific cd3-epsilon binding domain |
IL201340A IL201340A (en) | 2007-04-03 | 2009-10-01 | Cross-species-specific cd3- epsilon binding antibody |
ZA2009/07117A ZA200907117B (en) | 2007-04-03 | 2009-10-13 | Cross-species-specific cd3-epsilon binding domain |
HK10107648.9A HK1141300A1 (en) | 2007-04-03 | 2010-08-10 | Cross-species-specific cd3-epsilon binding domain |
HRP20130883TT HRP20130883T4 (en) | 2007-04-03 | 2013-09-18 | Cross-species-specific cd3-epsilon binding domain |
IL234081A IL234081B (en) | 2007-04-03 | 2014-08-12 | Cross - species - specific cd3 - epsilon binding antibody |
CY181101157T CY1120822T1 (en) | 2007-04-03 | 2018-11-05 | SPECIAL SPECIFIC COMMITMENT TERRITORY |
US18/066,972 US20230357444A1 (en) | 2007-04-03 | 2022-12-15 | Cross-species-specific binding domain |
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US18/066,972 Continuation-In-Part US20230357444A1 (en) | 2007-04-03 | 2022-12-15 | Cross-species-specific binding domain |
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JP (1) | JP2010524851A (en) |
KR (2) | KR101940944B1 (en) |
CN (2) | CN103694350B (en) |
CY (1) | CY1120822T1 (en) |
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FI (1) | FI2155783T4 (en) |
HR (1) | HRP20181902T1 (en) |
IL (1) | IL201340A (en) |
LT (1) | LT2520590T (en) |
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NZ (1) | NZ580755A (en) |
PL (2) | PL2155783T5 (en) |
PT (1) | PT2155783E (en) |
RS (2) | RS53008B2 (en) |
RU (1) | RU2561457C2 (en) |
SG (3) | SG182234A1 (en) |
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