NZ618021B2 - Cd3-binding molecules capable of binding to human and non-human cd3 - Google Patents
Cd3-binding molecules capable of binding to human and non-human cd3 Download PDFInfo
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- NZ618021B2 NZ618021B2 NZ618021A NZ61802112A NZ618021B2 NZ 618021 B2 NZ618021 B2 NZ 618021B2 NZ 618021 A NZ618021 A NZ 618021A NZ 61802112 A NZ61802112 A NZ 61802112A NZ 618021 B2 NZ618021 B2 NZ 618021B2
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Abstract
Disclosed is a CD3-binding molecule comprising an antigen-binding fragment of an antibody, wherein the antigen-binding fragment comprises an antibody CD3-specific VL domain and an antibody CD3-specific VH domain, wherein the CD3-specific VL domain and the CD3-specific VH domain form an antigen-binding domain capable of immunospecifically binding to both an epitope of human CD3 and to an epitope of the CD3 of a non-human mammal, wherein the VH and VL chains are of the sequences as defined in the specification. ng domain capable of immunospecifically binding to both an epitope of human CD3 and to an epitope of the CD3 of a non-human mammal, wherein the VH and VL chains are of the sequences as defined in the specification.
Description
recogn1zed by the system as De1ng Iore1gn to the body. ln st, the cellular
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Title of the Invention:
CD3-Binding Molecules Capable of g to
Human and man CD3
Cross-Reference to Related Applications:
This application claims priority to United States Patent Applications Nos.
61/488,716 (filed on May 21, 2011; pending) and 61/530,353 (filed on September 1,
2011; pending), each of which applications is herein incorporated by reference in its
entirety.
Reference to Sequence Listing:
This application includes one or more Sequence Listings pursuant to 37
C.F.R. 1.821 et seq., which are disclosed in both paper and computer-readable media,
and which paper and computer-readable disclosures are herein incorporated by
reference in their entirety.
ound of the Invention:
Field of the ion:
The present invention relates to CD3-binding molecules capable of binding
to human and non-human CD3, and in particular to such molecules that are cross-
reactive with CD3 of a non-human mammal (e.g., a cynomolgus monkey). The
invention also pertains to uses of such antibodies and antigen-binding fragments in the
treatment of cancer, autoimmune and/or inflammatory diseases and other conditions.
Description of Related Art:
The body's immune system serves as a defense against a variety of
conditions, including, e.g., injury, ion and sia, and is mediated by two
separate but interrelated systems: the cellular and l immune systems.
Generally speaking, the humoral system is mediated by soluble products odies
or immunoglobulins) that have the ability to combine with and neutralize products
ized by the system as being foreign to the body. In contrast, the cellular
immune system involves the mobilization of certain cells, termed T cells, that serve a
Lnnnnnnyununubnyw; Lvuyunnuv uv yvavavLLu MAL“ Lu; ‘4;be uwvuuvuxvvu Lvuvu VAL v" u
characteristics: the ite specificity of the immune response for antigen
variety of therapeutic roles. T cells are lymphocytes that are derived from the thymus
and ate between the tissues, lymphatic system and the circulatory system. They
act t, or in response to, a variety of foreign structures (antigens). In many
instances these foreign antigens are expressed on host cells as a result of neoplasia or
infection. Although T cells do not themselves secrete antibodies, they are usually
required for antibody secretion by the second class of lymphocytes, B cells (which
derive from bone marrow). Critically, T cells t extraordinary immunological
specificity so as to be capable of discerning one n from another).
A naive T cell, e.g., a T cell which has not yet encountered its specific
antigen, is activated when it first encounters a specific peptide:MHC complex on an
antigen presenting cell. The antigen presenting cell may be a B cell, a hage or
a dendritic cell. When a naive T cell encounters a specific peptide:MHC complex on
an n presenting cell, a signal is delivered through the T-cell receptor which
induces a change in the conformation of the T cell’s lymphocyte function associated
n (LFA) molecules, and increases their affinity for intercellular adhesion
molecules (ICAMs) present on the surface of the antigen presenting cell. The signal
generated by the ction of the T cell with an antigen presenting cell is necessary,
but not sufficient, to activate a naive T cell. A second co-stimulatory signal is
required. The naive T cell can be activated only by an antigen-presenting cell
carrying both a specific peptide MHC complex and a co-stimulatory molecule on its
surface. Antigen recognition by a naive T cell in the absence of co-stimulation results
in the T cell becoming anergic. The need for two signals to activate T cells and B
cells such that they achieve an adaptive immune response may e a mechanism
for avoiding responses to self-antigens that may be present on an antigen presenting
cell at locations in the system where it can be recognized by a T cell. Where contact
of a T cell with an n presenting cell s in the generation of only one of two
required s, the T cell does not become activated and an ve immune
response does not occur.
The efficiency with which humans and other mammals develop an
immunological se to pathogens and foreign substances rests on two
characteristics: the exquisite specificity of the immune response for antigen
complex has such a large number of ITAMS (10 in all), and these ITAMS are arrayed
in tandem (due to the dimerization of the tuent chains), phosphorylation of the
recognition, and the immunological memory that allows for faster and more Vigorous
responses upon re-activation with the same n (Portoles, P. et al. (2009) “The
3 Complex: Opening the Gate to sful Vaccination,” Current
Pharmaceutical Design 15:3290-3300; Guy, C.S. et al. (2009) ization of
Proximal Signal Initiation at the TCR:CD3 Complex,” Immunol ReV. 232(l):7-2l).
The city of the response of T-cells is mediated by the recognition of antigen
(displayed on Antigen-Presenting Cells (APCs) by a molecular complex involving the
T Cell Receptor ) and the cell surface receptor ligand, CD3. The TCR is a
covalently linked heterodimer of or and B chains (“TCRaB”). These chains are class I
membrane polypeptides of 259 (or) and 296 ([3) amino acids in length. The CD3
molecule is a complex containing a CD3 y chain, a CD3 5 chain, and two CD3 8
chains associated as three dimers (83/, 88, CC) (Guy, C.S. et al. (2009) “Organization of
al Signal Initiation at the TCR:CD3 x,” Immunol ReV. :7-2l;
Call, M.E. et al. (2007) “Common Themes In The Assembly And Architecture Of
Activating Immune Receptors,” Nat. Rev. Immunol. 7:841-850; Weiss, A. (1993) “T
Cell Antigen Receptor Signal Transduction: A Tale Of Tails And Cytoplasmic
Protein-Tyrosine Kinases,” Cell 73:209-2l2). The TCR and CD3 complex, along
with the CD3 5; chain zeta chain (also known as T-cell receptor T3 zeta chain or
CD247) comprise the TCR complex (van der Merwe, P.A. etc. (epub Dec. 3, 2010)
“Mechanisms For T Cell Receptor ring,” Nat. ReV. Immunol. 11:47-55;
Wucherpfennig, KW. et al. (2010) “Structural Biology of the T-cell Receptor:
Insights into Receptor Assembly, Ligand Recognition, and Initiation of Signaling,”
Cold Spring Harb. Perspect. Biol. 2:a005140). The complex is particularly significant
since it contains a large number (ten) of immunoreceptor tyrosine-based activation
motifs (ITAMs).
In mature T cells, TClVCD3 activation by foreign antigenic peptides
associated to self-MHC molecules is the first step needed for the ion of
antigen-specific T cells, and their differentiation into effector or memory T
lymphocytes. These processes involve the phosphorylation of the immunoreceptor
tyrosine-based activation motifs (ITAMs) of the TCR complex. Because the TCR
complex has such a large number of ITAMS (10 in all), and these ITAMS are d
in tandem (due to the dimerization of the constituent chains), phosphorylation of the
vuvv Allvllvuvlvuuuvt/t/I vuquV Avarvv, ALwLLuyLwLLuwuLuLL IA.:./v 1.2.2, L'ALuvvvuv, L‘. V» w!»-
(2003) “Individualized T Cell Monitored Administration Of ATG Versus OKT3 In
relevant tyrosine residues upon TCR ligation creates paired docking sites for ns
that contain Src homology 2 (SH2) domains such as the f; chain-associated protein of
70 kDa (ZAP-70), and thereby initiate an amplifying signaling cascade which leads to
T-cell activation and differentiation (Guy, C.S. et al. (2009) “Organization of
Proximal Signal Initiation at the TCR.'CD3 Complex,” Immunol Rev. 232(1):7-2l).
The outcome of these processes is ted by the intensity and quality of
the antigen stimulus, as well as by the nature of accompanying signals delivered by
co-receptor and co-stimulatory surface molecules, or by cytokine receptors (Portoles,
P. et al. (2009) “The TCR/CD3 Complex.‘ Opening the Gate to Successful
Vaccination,” Current Pharmaceutical Design 15:3290-3300; Riha, P. et al. (2010)
“CD28 Co-Signaling In The Adaptive Immune Response,” Self/Nonself l(3):23l-
240). Although TCR stimulation is a prerequisite for T-cell activation, it is well
recognized that engagement of co-stimulatory molecules, such as CD28, is necessary
for filll T-cell activation and differentiation (Guy, C.S. et al. (2009) ization of
Proximal Signal Initiation at the D3 Complex,” Immunol Rev. 232(1):7-2l).
Due to the fundamental nature of CD3 in initiating an anti-antigen response,
monoclonal antibodies against this receptor have been proposed as being capable of
blocking or at least modulating the immune process and thus as agents for the
treatment of inflammatory and/or autoimmune disease. Indeed, anti-CD3 dies
were the first antibody approved for the human therapy (St. Clair E.W. (2009) “Novel
Targeted Therapies for Autoimmunity,” Curr. Opin. Immunol. 648-657). Anti-
CD3 antibody (marketed as LONE TM OKT3TM by Janssen-Cilag) has been
administered to reduce acute rejection in ts with organ transplants and as a
treatment for lymphoblastic ia (Cosimi, AB. et al. (1981) “Use Of
Monoclonal dies To T-Cell Subsets For Immunologic Monitoring And
Treatment In Reczpients OfRenal Allografts,” N. Engl. J. Med. 8-314; Kung,
P. et al. (1979) Monoclonal antibodies defining distinctive human T cell surface
antigens,” Science 7-349; Vigeral, P. et al. (1986) “Prophylactic Use OfOKT3
Monoclonal Antibody In Cadaver Kidney Reczpients. Utilization Of OKT3 As The
Sole Immunosuppressive Agent,” lantation 41:730-733; Midtvedt, K. et al.
(2003) idualized T Cell Monitored Administration Of ATG Versus OKT3 In
Interleukin-2, And Gamma-Interferon In Serum After Injection 0f OKT3 Monoclonal
Antibody In Kidney Transplant Recipients.” Transplantation 47:606-608; Ferran. C. et
Steroid-Resistant Kidney Graft Rejection,” Clin. Transplant. 17(1):69-74; Gramatzki,
M. et al. (1995) “Therapy With 0KT3 Monoclonal Antibody In Refractory T Cell
Acute Lymphoblastic Leukemia Induces Interleukin-2 Responsiveness,” Leukemia
9(3):382-390; Herold, K.C. et al. (2002) “Anti-CD3 Monoclonal Antibody In New-
Onset Type I Diabetes Mellitus,” N. Engl. J. Med. 346:1692-1698; Cole, M.S. et al.
(1997) “Human IgG2 Variants 0f ic Anti-CD3 Are Nonmitogenic to T cells,”
J. Immunol. 159(7):3613-3621; Cole, M.S. et al. (1999) “I-Ium291, A Humanized
Anti-CD3 Antibody, Is Immunosuppressive To T Cells While ting Reduced
Mitogenicity in vitro,” lantation 68:563-571; US. Patent Nos. 6,491,916;
,585,097 and 6,706,265).
r, such anti-CD3 treatment has not proven to be specific enough to
avoid side effects gsson, J. (2009) “The Role nomodulation Therapy in
Autoimmune Diabetes,” J. Diabetes Sci. Technol. 3(2):320-330). Repeated daily
administration of OKT3 results in profound suppression and provides
effective treatment of rejection following renal transplantation. The in vivo
administration of OKT3 results in both T cell activation and suppression of immune
responses. However, the use of OKT3 has been hampered by a first toxic dose
reaction syndrome that is related to initial T-cell activation events and to the ensuing
release of cytokines that occurs before immunosuppression of T cell responses. The
ed side effects that follow the first and sometimes the second injection of this
mouse monoclonal antibody include a “flu-like” syndrome consisting of high fever,
, headache, and gastrointestinal symptoms (vomiting and diarrhea) and in severe
cases pulmonary edema within hours of treatment has been noted (Thistlethwaite, J.R.
Jr. et al. (1988) “Complications and Monitoring of 0KT3 y,” Am. J. Kidney
Dis. 11:112-119). This syndrome is believed to reflect OKT3-mediated cross-linking
of the TCIVCD3 complex on the T cell surface and the resultant release of cytokines
(e.g, tumor necrosis factor alpha (TNFu), interferon-y, interleukins IL-2, IL-3, IL-4,
IL-6, IL-10 and granulocyte-macrophage colony-stimulating factor (Masharani, U.B.
et al. (2010) “Teplizumab y For Type I Diabetes,” Expert Opin. Biol. Ther.
(3):459-465; Abramowicz, D. et al. (1989) “Release 0f Tumor Necrosis Factor,
Interleukin-2, And Gamma-Interferon In Serum After ion 0f OKT3 Monoclonal
Antibody In Kidney lant Recipients,” Transplantation -608; Ferran, C. et
above. Therefore, preclinical data generated in rodents are of d predictive
“A-“A“ "724.1, «AMAAAL LA LLA 4...”. nn«,;l.‘,;ln4.,\ 'T‘LA ..,.,\n,.L AL‘ ALAIAA 13,“. "ADAA.-. LANL£«,. In
al. (1990) “Cytokine-Related Syndrome Following Injection 0fAnti—CD3 Monoclonal
Antibody: Further ce For Transient In Vivo T Cell Activation,” Eur. J.
Immunol. 20:509-515; Hirsch, R. et al. (12989) “Eflects OfIn Vivo Administration 0f
Anti-CD3 Monoclonal Antibody 0n T Cell Function In Mice. II. In Vivo Activation Of
T Cells,” J. l. 142:737-743). The use of anti-CD3 antibodies is disclosed in
United States Patents Nos. 7,883,703; 7,728,114; 472; 7,575,923; and
7,381,903, and in United States Patent Publications Nos. 2010/0150918;
2010/0209437; 2010/0183554; 2010/0015142, 2008/0095766, 077246 and in
PCT Publication /119567.
A particular limitation of prior antibodies is their specificity for only human
CD3. This limitation is a significant impediment to the pment of such
antibodies as therapeutic agents for the ent of human diseases. In order to
obtain market approval any new candidate medication must pass through rigorous
testing. This testing can be subdivided into preclinical and al phases. Whereas
the latter — filrther subdivided into the generally known clinical phases 1, II and III — is
performed in human patients, the former is performed in animals. The aim of preclinical
testing is to prove that the drug candidate has the desired activity and most
importantly is safe. Only when the safety in animals and possible effectiveness of the
drug candidate has been ished in nical testing this drug candidate will be
approved for clinical testing in humans by the respective regulatory authority. Drug
candidates can be tested for safety in animals in the following three ways, (i) in a
relevant species, i.e., in 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 enic animals are that this technology is
typically limited to rodents. However, rodents and humans have significant
differences in logy that may complicate the extrapolation of safety data
obtained in s to predict safety in . 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. Therefore, preclinical data generated in rodents are of limited predictive
power with respect to the drug candidate. The approach of choice for safety testing is
group consisting of h-mab2 VH-l (SEQ ID NO:36), h-mab2 VH-2 (SEQ ID
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 cynomolgus monkeys.
ingly, an anti-CD3 antibody capable of binding to both human and primate
CD3 is highly desirable. Such antibodies have been described in United States Patent
Publication No. 20100150918 and in PCT Publication /119567.
Despite such es, a need remains for anti-human CD3 antibodies and
their antigen-binding fragments that are capable of cross-reacting with CD3 of a non-
human mammal (e.g., a cynomolgous monkey). The present invention addresses this
need and the need for ed therapeutics for cancer, autoimmunity and
inflammatory diseases.
y of the Invention:
The present invention relates to CD3-binding molecules capable of g
to human and man CD3, and in particular to such molecules that are cross-
reactive with CD3 of a non-human mammal (e.g., a lgus monkey). The
invention also pertains to uses of such antibodies and antigen-binding fragments in the
treatment of cancer, autoimmune and/or inflammatory es and other conditions.
In detail, the invention provides a CD3-binding molecule comprising an
antigen-binding nt of an antibody, wherein the antigen-binding fragment
comprises an antibody CD3-specific VL domain and an antibody ecific VH
domain, wherein the CD3-specific VL domain and the CD3-specific VH domain form
an antigen-binding domain capable of immunospecif1cally binding to both an epitope
of human CD3 and to an epitope of the CD3 of a non-human mammal, wherein:
(I) the CD3-specific VL domain is selected from the group consisting of h-mab2
VL-1 (SEQ ID NO:16), h-mab2 VL-2 (SEQ ID NO:18), h-mab2 VL-3 (SEQ
ID NO:20), h-mab2 VL-4 (SEQ ID , h-mab2 VL-S (SEQ ID NO:24),
h-mab2 VL-6 (SEQ ID NO:26), h-mab2 VL-7 (SEQ ID NO:28), h-mab2
VL-8 (SEQ ID NO:30), h-mab2 VL-9 (SEQ ID NO:32), and h-mab2 VL-10
(SEQ ID NO:34), and said CD3-specific VH domain is selected from the
group consisting of h-mab2 VH-l (SEQ ID NO:36), h-mab2 VH-2 (SEQ ID
[CITIIIIIUS 'dIILl II'Ol'II 1V -[CI'1'IIIIIUS [O L-[CITIIIIIUSI
(i) a domain (A) comprising the CD3-specif1c VL domain;
NO:38), h-mab2 VH-3 (SEQ ID NO:40), h-mab2 VH-4 (SEQ ID , h-
mab2 VH-S (SEQ ID NO:44), h-mab2 VH-6 (SEQ ID NO:46), h-mab2 VH-
6L (SEQ ID NO:54), h-mab2 VH-7 (SEQ ID NO:48), h-mab2 VH-8 (SEQ
ID NO:50), h-mab2 VH-8L (SEQ ID NO:55), h-mab2 VH-8 di-l (SEQ ID
NO:56), h-mab2 VH-8 di-2 (SEQ ID NO:57), h-mab2 VH-6M (SEQ ID
NO:72), h-mab2 VH-8M (SEQ ID NO:74), h-mab2 VH-Zk (SEQ ID
NO:87), and h-mab2 VH-Sk (SEQ ID NO:88); or
(II) the CD3-specif1c VL domain is selected from the group consisting of h-mabl
VL-l (SEQ ID NO:10) and h-mabl VL-2 (SEQ ID NO:12), and the CD3-
specific VH domain is h-mabl VH (SEQ ID NO:14).
The invention particularly concerns the embodiment of the above-described
CD3-binding molecule wherein the ecif1c VL domain is h-mab2 VL-6 (SEQ
ID NO:26).
The ion further concerns the embodiment of the above-described CD3-
binding molecules wherein the CD3-specif1c VH domain is h-mab2 VH-8 (SEQ ID
NO:50), h-mab2 VH-6 (SEQ ID NO:46), or h-mab2 VH-Zk (SEQ ID NO:87).
The invention ularly concerns the embodiment of the above-described
CD3-binding molecule wherein the molecule is an antibody, and particularly, wherein
the antibody lacks an Fc region or ses an Fc region that:
(A) lacks or fianction or has reduced effector fianction; or
(B) s the ability of the Fc region of the antibody to bind to an Fc receptor;
wherein the reduction in or function and the impairment of binding ability is
ve to that of a wild-type Fc receptor.
The invention r concerns the embodiment of the above-described CD3-
binding molecules wherein the molecule is a CD3-binding diabody that comprises a
first polypeptide chain and a second polypeptide chain, the chains being covalently
bonded to one another, wherein:
I. the first polypeptide chain comprises an amino us and a carboxy
terminus and from N-terminus to C-terminus:
(i) a domain (A) comprising the CD3-specif1c VL domain;
(ii) a domain (B) comprising a binding region of a heavy chain variable
domain of a second immunoglobulin (VH2); and
(iii) a domain (C);
wherein the domains (A) and (B) do not associate with one another to form an
epitope binding site;
(H) the second ptide chain comprises an amino terminus and a carboxy
terminus and from N-terminus to C-terminus:
(i) a domain (D) comprising a binding region of a light chain variable
domain of the second immunoglobulin (VL2);
(ii) a domain (E) comprising the CD3-specific VH domain; and
(iii) a domain (F);
wherein the domains (D) and (E) do not associate with one another to form an
epitope binding site; and
wherein:
(1) the domains (A) and (E) associate to form the antigen-binding domain that is
capable of immunospecif1cally binding to both human CD3 and to the CD3 of
a non-human ;
(2) the domains (B) and (D) associate to form a binding site that
immunospecifically binds to a second epitope, the second epitope being
different from the CD3 epitope bound by the antigen-binding domain formed
from the association of the domains (A) and (E); and
(3) the domains (C) and (F) are covalently associated together.
The invention further concerns the embodiment of the above-described CD3-
binding les n the second epitope is not an epitope of CD3.
The invention further concerns the embodiment of the above-described CD3-
binding molecules wherein the second e is an epitope of CD3 that is different
from the CD3 epitope bound by the antigen-binding domain formed from the
association of the domains (A) and (E).
association and the molecule involved in the T cell — B cell association is selected
The invention further concerns the embodiment of the above-described CD3-
binding molecules or antibodies or diabodies in which such le humanized.
The ion further concerns the embodiment of the above-described CD3-
binding molecules or antibodies or diabodies in which such molecule is capable of
immunospecifically binding to CD3 and to fluorescein.
The invention further concerns the embodiment of the above-described CD3-
binding molecules or diabodies in which such molecule is capable of
immunospecif1cally binding to both: (i) CD3 and (ii)(a) a tumor antigen, or (ii)(b) a
cell surface antigen, receptor or receptor ligand.
The invention further concerns the embodiment of the above-described CD3-
binding molecules or ies in which the molecule or diabody is capable of
immunospecif1cally binding to CD3 and to a tumor antigen expressed on a tumor cell,
wherein the tumor cell is a tumor cell from a cancer selected from the group
consisting of: breast cancer, prostate , c cancer, lung cancer, stomach
cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, ovarian cancer,
oral cavity , pharyngeal cancer, esophageal cancer, laryngeal cancer, bone
cancer, skin cancer, melanoma, uterine cancer, testicular cancer, bladder cancer,
kidney cancer, brain cancer, glioblastoma, thyroid cancer, lymphoma, myeloma, and
leukemia.
The invention further concerns the embodiment of the above-described CD3-
g molecules or diabodies in which the molecule or diabody is capable of
specif1cally g to CD3 and to a cell surface antigen, receptor or receptor
ligand, wherein the cell surface antigen, receptor or receptor ligand is HER2/neu, B7-
H3, CD20, PSMA, IGF-lR., Ep-CAM, or is a molecule involved in a T cell — B cell
association that leads to T cell or B cell activation in an adaptive immune se.
The invention further concerns the embodiment of the described CD3-
binding molecules or ies in which the le or diabody is e of
immunospecif1cally binding to CD3 and to a molecule involved in the T cell — B cell
association and the molecule involved in the T cell — B cell association is selected
from the group consisting of CD19, CD20, CD22, CD23, CD27, CD32B, CD38,
CD40, CD79a, CD79b, CD80, CD86, LFA-I, LFA-3 and CFA-I.
The invention further concerns a pharmaceutical ition comprising
any of the above-descibed CD3-binding molecules, antibodies or diabodies, and a
pharmaceutically acceptable carrier, excipient or diluent.
The invention further concerns the above-described pharmaceutical
ition for use in the treatment of cancer or an autoimmune or inflammatory
disease.
The invention further concerns the above-described pharmaceutical
composition for use in the treatment of an autoimmune or inflammatory disease
selected from the group ting of: type I insulin-dependent diabetes, rheumatoid
arthritis, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel
disease, myasthenia gravis, celiac’s disease, Sjogren's syndrome, Grave’s e,
Crohn’s disease, autoimmune tis, psoriasis, psoriatic arthritis, asthma, allergic
rhinitis, effects from organ transplantation, or graft vs. host disease (GVHD). The
invention particularly concerns the above-described pharmaceutical composition for
use in the treatment of type I insulin-dependent diabetes.
Brief Description of the Drawings:
Figures lA-lB show the results of a capture ELISA in which the ability of
anti-CD3 antibody mABl (Figure 1A) or a chimeric derivative of antibody mABl
(ch-mAbl) (Figure 1B) was assessed using human soluble CD3 3”).
Figures 2A-2B show the results of a capture ELISA in which the ability of
anti-CD3 antibody mAB2 (Figure 2A) or a chimeric derivative of dy mAB2
(ch-mAb2) (Figure 2B) was ed using human soluble CD3 (“shCD3”) or soluble
cynomolgus monkey CD3 (“scCD3”).
Figure 3 show the s of es to determine the effect of variations in
Kabat numbered framework residues 41-46 of the light chain ofmAb2.
1)’ lllpllulllu UVLLD’ 1' [Sui b 1.)”. 1 Mull VULVU 1\1111115 UL J V1\U_ 1 llulllull illullLLV UVLL
lymphoma cells
Figure 4 show the s of analyses to determine the effect of variations in
Kabat numbered framework residues 36, 38, 44 and 46 of the light chain ofmAb2.
Figure 5 show the results of es to determine the effect of variations in
Kabat numbered framework es 36, 38 and 46 of the light chain .
Figure 6 show the results of analyses to determine the effect of variations in
Kabat numbered framework residues 30, 49 and 93 of the heavy chain ofmAb2.
Figure 7 show the results of additional es conducted to determine the
effect of variations in Kabat numbered framework residues 30, 49 and 93 of the heavy
chain of mAb2.
Figures 8A-8B show the results of analyses conducted to assess the ability
of chimeric and humanized mAb2 to bind to non-human CD3.
Figures 9A-9D show gram tracings of BIACORETM analyses done to
determine the kinetics of the binding of ch-mAB2 or h-mAb2 and scCD3 or scCD3.
Figures 10A-10D show the results of capture ELISAs performed on
DARTTM ies having an anti-CD3 first epitope binding site and second epitope
binding site that bind to either Her2/neu, CD19, EGFR, or B7-H3.
Figures 11A-11B show the ability of B7H3 x CD3 DARTTM diabodies to
mediate redirected killing of tumor cells expressing B7H3.
Figures 12A-12E show the ability of A33 x CD3 DARTTM diabodies to
mediate redirected killing of tumor cells expressing A33.
Figures 13A and 133 show the results of a comparison of the capacity of a
CD19-h-mAb2 DARTTM and a CD19 x CD3 DART diabody to cause redirected T-
ediated killing. The CD19-h-mAb2 DARTTM diabody exhibits specificity to
human as well as non-human CD3; the CD19 x CD3 DART diabody o exhibits
specificity only to human CD3. Figure 13A: redirected g of Raji human B-cell
lymphoma cells; Figure 13B: redirected killing of JeKo-l human mantle cell
lymphoma cells
antigen-binding domain exhibits reactivity such that it will immunospecif1cally
Figures 14A and 143 show that the CD19-h-mAb2 DARTTM diabody of the
present invention was able to e sis in the presence of either human or
non-human T-cell effector cells.
Figures 15A and ISB show the ability of the ERBITUXTM-h-mAbZ
DARTTM diabody of the present invention or an ERBITUXTM-T-Cell Receptor
DARTTM y to mediate an increase in CD69 MFI upon incubation with CD4+ or
CD8+ T cells; A control ERBITUXTM-FN18 CD3 DARTTM diabody (capable of
binding to EGFR and to cynolmolgus monkey CD3) failed to induce an increase in
the CD69 MFI.
Figures 16A-16D show the results of investigations into the binding of
either ERBITUXTM-h-mAbZ DARTTM diabody, ERBITUXTM-m-mAbZ DARTTM
diabody or 4420-h-mAb2 DARTTM diabody ive control) or a l secondary to
A498 or A431 cells (Figure 16A and 16C, tively), and to mediate redirected
killing of such cells (Figure 16B and 16D, respectively).
Detailed Description of the Invention:
The present invention relates to anti-human CD3 antibodies and their
antigen-binding fragments, and in particular to such antibodies that are cross-reactive
with CD3 of a non-human mammal (e.g., a cynomolgous monkey). The invention
also pertains to uses of such antibodies and antigen-binding nts in the treatment
of cancer, autoimmune and/or inflammatory diseases and other conditions.
1. ions
As used herein, the term “CD3-binding molecule” denotes a molecule
capable of immunospecific binding to both human CD3 and to the CD3 of a non-
human mammal through at least one antigen recognition site (e.g., an antigen-binding
domain of an antibody) located in the variable region of the molecule. As used herein
such lity to immunospecifically bind to both human CD3 and to the CD3 of a
man mammal is not intended to denote a capacity of a single antigen binding
domain to simultaneously bind to both such CD3 molecules, but rather that such an
antigen-binding domain exhibits cross-reactivity such that it will immunospecif1cally
v “LLWVLV vanunn u; w unnnvnvnnu \v.b., ULLV UVUVLLu/ “A AL‘A “LWVVuJ FunjtjvtjunuLLLLLLLLLu
form an epitope binding site. DARTTM ies may be monospecific, bispecif1c,
bind to human CD3 when incubated in the presence of human CD3 and will
immunospecif1cally bind to the CD3 of a non-human mammal when incubated in the
presence of such non-human mammalian CD3.
As used , the term inding molecule” asses not only
intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab,
Fab', F(ab')2 FV), single chain (ScFV), mutants thereof, naturally occurring variants,
fusion proteins comprising an antibody n with an antigen ition site of the
ed specificity, humanized antibodies, chimeric antibodies, “BiTEs®,”
“DARTTM” diabody molecules and any other modified configuration of the
immunoglobulin molecule that comprises an antigen ition site of the required
specificity. The term “BiTEs” (bi-specific T-cell engagers) refers to a single
polypeptide chain molecule that haVing two antigen-binding domains, one of which
binds to a T-cell antigen and the second of which binds to an antigen present on the
surface of a target ( WO 547; Baeuerle, P et al. (2008) “BiTE®.' A New Class
OfAntibodies That Recruit T Cells,” Drugs of the Future 33: 137-147; , et al.
2008) “Tumor Regression in Cancer ts by Very Low Doses of a T Cell-
Engaging Antibody,” Science 321: 974-977).
The term “DARTTM” (Dual Affinity ReTargeting reagent) y refers to
an immunoglobulin le that comprises at least two polypeptide chains that
associate (especially through a covalent interaction) to form at least two epitope
binding sites, which may recognize the same or different epitopes. Each of the
polypeptide chains of a DARTTM diabody se an immunoglobulin light chain
variable region and an immunoglobulin heavy chain variable region, but these regions
do not interact to form an epitope binding site. Rather, the immunoglobulin heavy
chain variable region of one (e.g, the first) of the DARTTM diabody polypeptide
chains interacts with the immunoglobulin light chain variable region of a different
(e.g., the second) DARTTM polypeptide chain to form an epitope binding site.
Similarly, the immunoglobulin light chain variable region of one (e.g, the first) of the
DARTTM diabody polypeptide chains interacts with the immunoglobulin heavy chain
variable region of a different (e.g., the second) DARTTM diabody polypeptide chain to
form an epitope binding site. DARTTM diabodies may be monospecific, bispecif1c,
rcinoma antigen, LEA, lung adenocarcinoma F3 antigen, malignant human
lvmphocvte antigen-APO-l. melanoma antigen gn75. melanoma-associated antigen
trispecific, etc., thus being able to simultaneously bind one, two, three or more
different epitopes (which may be of the same or of different antigens). DARTTM
diabodies may additionally be lent, bivalent, trivalent, tetravalent,
pentavalent, hexavelent, etc., thus being able to simultaneously bind one, two, three,
four, five, six or more molecules. These two attributes of DARTTM diabodies ,
degree of specificity and valency may be combined, for example to produce if1c
antibodies (i.e., capable of binding two epitopes) that are tetravalent (i.e., capable of
binding four sets of epitopes), etc. DARTTM diabody molecules are disclosed in PCT
Publications WO 13665, , and .
The bispecific (or trispecific or multispecif1c) molecules of the present
invention will be capable of binding to both human CD3 and the CD3 of a non-human
mammal (e.g., lgous monkey), and also to a second (or additional) and
different n(s) or epitope(s). The second antigen or epitope is preferably a tumor
antigen expressed on a tumor cell. Such tumor cells may be from cancers, for
example, breast cancer, prostate cancer, gastric cancer, lung cancer, h cancer,
colon cancer, rectal cancer, pancreatic cancer, liver cancer, n cancer, oral cavity
cancer, pharyngeal cancer, esophageal cancer, laryngeal cancer, bone cancer, skin
cancer, melanoma, uterine , testicular cancer, bladder cancer, kidney cancer,
brain cancer, astoma, thyroid cancer, lymphoma, myeloma, or leukemia. The
additional antigens or es are preferably cell surface tumor antigens or epitopes
(such as: 17-1A, A33, adult erythrocyte primary endoderm I antigen, alpha
fetoprotein, an envelope antigen of an RNA tumor virus, r tumor oncofetal
antigen, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, Burkitt’s lymphoma antigen-
38.13, CA125, CD18, CD19, human B-lymphoma antigen-CD20, CD22, CD33,
CD44, CD52, CEA, COl7-1A, CTA-1, CTLA-4, epidermal growth factor receptor,
Ep-CAM, EphA2, fetal erythrocyte I antigen, f1brosarcoma antigen, ganglioside GD2,
ganglioside GD3, ganglioside GM2, ganglioside GM3, GICA 19-9, gp IIIb/IIIa, gp72,
HERl, neu, HER3, HER4, high molecular weight melanoma antigen, HLA-
DR antigen, human leukemia T cell antigen-Gp37, human lung carcinoma antigen
L20, human lung carcinoma n L6, human milk fat globule antigen, IgE, KS 1/4
pan-carcinoma antigen, LEA, lung adenocarcinoma F3 antigen, malignant human
lymphocyte n-APO-l, melanoma antigen gp75, melanoma-associated antigen
immunoglobulins as well as the fragments etc. described above under the definition of
“onh‘knr‘xr ”
p97, neoglycoprotein, nuC242, polymorphic epithelial mucin antigen, prostate
specific antigen, prostate specific membrane antigen, prostatic acid phosphate, SK-l
n, TAG-72, T-antigen, tumor n CAlZS, tumor antigen MUCl, tumor-
specific transplantation type of cell-surface antigen, vascular endothelial growth
, vascular endothelial growth factor-receptor, and (va3). Alternatively, such
additional antigens or epitopes may be associated with a pathogen (such as: hepatitis
type A, hepatitis type B, hepatitis type C, influenza, lla, adenovirus, herpes
simplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirus,
echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus,
galovirus, echinovirus, arbovirus, huntavirus, coxsackie virus, mumps virus,
measles virus, rubella virus, polio virus, small pox, Epstein Barr virus, human
immunodeficiency virus type I (HIV-I), human immunodeficiency virus type 11 (HIVII
), viral miningitis, viral encephalitis, dengue, small pox; mycobacteria rickettsia,
mycoplasma, neisseria, S. pneumonia, Borrelia burgclorferi, us anthracis,
Streptococcus, Staphylococcus, Mycobacterium, tetanus, pertissus, cholera, plague,
diptheria, chlamydia, and legionella; leishmania, kokzidioa, trypanosoma or malaria;
chlamydia and tsia.
The term “monoclonal antibody” refers to a homogeneous antibody
population wherein the onal antibody is comprised of amino acids (naturally
ing and non- naturally occurring) that are involved in the selective binding of
an antigen. Monoclonal antibodies are highly specific, being directed against a single
antigenic site. The term “monoclonal antibody” encompasses not only intact
monoclonal antibodies and filll- length monoclonal antibodies, but also nts
thereof (such as Fab, Fab', F(ab')2 Fv), single chain (ScFv), mutants thereof, fusion
proteins sing an antibody portion, humanized monoclonal antibodies, chimeric
monoclonal antibodies, and any other modified ration of the immunoglobulin
molecule that ses an antigen ition site of the required specificity and the
ability to bind to an antigen. It is not intended to be limited as regards to the source of
the antibody or the manner in which it is made (e. g., by oma, phage selection,
recombinant expression, transgenic animals, etc.). The term includes whole
immunoglobulins as well as the fragments etc. described above under the definition of
“antibody.”
88:4181-4185; Tempest, P.R. et al. (1991) “Reshaping A Human Monoclonal
The term “humanized antibody” refer to a chimeric molecule, generally
prepared using recombinant techniques, having an antigen binding site d from
an immunoglobulin from a non-human species and the remaining immunoglobulin
structure of the le based upon the structure and /or sequence of a human
immunoglobulin. The antigen-binding site may comprise either te variable
domains fused onto nt domains or only the complementarity ining
regions (CDRs) grafted onto appropriate framework regions in the variable domains.
Antigen binding sites may be wild type or modified by one or more amino acid
substitutions. This eliminates the constant region as an immunogen in human
individuals, but the possibility of an immune response to the foreign variable region
remains (LoBuglio, A.F. et al. (1989) “Mouse/Human Chimeric Monoclonal Antibody
In Man.‘ Kinetics And Immune Response,” Proc. Natl. Acad. Sci. (USA) 86:4220-
4224). r approach focuses not only on providing human-derived constant
regions, but modifying the variable regions as well so as to reshape them as closely as
possible to human form. It is known that the variable regions of both heavy and light
chains contain three complementarity- determining regions (CDRs) which vary in
response to the ns in on and ine binding capability, flanked by four
framework regions (FRs) which are relatively conserved in a given species and which
putatively provide scaffolding for the CDRs. When nonhuman antibodies are prepared
with t to a particular antigen, the variable regions can be “reshaped” or
“humanized” by grafting CDRs derived from nonhuman antibody on the FRs present
in the human antibody to be modified. Application of this approach to various
antibodies has been reported by Sato, K. et al. (1993) Cancer Res 53:851-856.
Riechmann, L. et al. (1988) ping Human Antibodies for Therapy,” Nature
332:323-327; Verhoeyen, M. et al. (1988) “Reshaping Human Antibodies: ng
An Antilysozyme Activity,” Science 239: 536; Kettleborough, C. A. et al. (1991)
“Humanization OfA Mouse Monoclonal Antibody By CDR-Grafting.‘ The Importance
OfFramework Residues 0n Loop Conformation,” Protein Engineering 4:773-3783;
Maeda, H. et al. (1991) “Construction Of Reshaped Human Antibodies With HIV-
Neutralizing Activity,” Human Antibodies Hybridoma 2: 124-134; Gorman, S. D. et al.
(1991) “Reshaping A Therapeutic CD4 dy,” Proc. Natl. Acad. Sci. (USA)
88:4181-4185; Tempest, P.R. et al. (1991) ping A Human Monoclonal
(e.g, an anti-CD3 antibody) to bind to the epitope under different conditions, for
Antibody To t Human Respiratory ial Virus Infection in vivo,”
Bio/Technology 9:266-271; Co, M. S. et al. (1991) “Hamanized Antibodies For
Antiviral Therapy,” Proc. Natl. Acad. Sci. (USA) 88:2869-2873; Carter, P. et al.
(1992) “Hamanization OfAn Anti-p185her2 dy For Human Cancer Therapy,”
Proc. Natl. Acad. Sci. (USA) 89:4285-4289; and Co, MS. et al. (1992) “Chimeric
And Hamanized Antibodies With Specificity For The CD33 Antigen,” J. Immunol.
148:1149-1154.
In some embodiments, humanized antibodies preserve all CDR sequences
(for example, a humanized mouse antibody which contains all six CDRs from the
mouse antibodies). In other embodiments, humanized dies have one or more
CDRs (one, two, three, four, five, six) which are altered with respect to the original
antibody, which are also termed one or more CDRs “derived from” one or more
CDRs from the al antibody. As disclosed below, the preferred antibodies of the
present invention have ic identified CDRs. The present invention, however,
contemplates equivalent antibodies having altered CDRs.
As used herein, an antibody or a polypeptide is said to “immunospecif1cally”
or equivalently, “specifically” bind a region of another molecule (i.e., an e) if it
reacts or associates more frequently, more rapidly, with greater duration and/or with
greater affinity with that e ve to ative epitopes. For example, an
antibody that specifically binds to a CD3 epitope is an antibody that binds this CD3
epitope with greater affinity, avidity, more readily, and /or with greater duration than
it binds to other CD3 epitopes or non-CD3 epitopes. It is also understood by reading
this tion that, for e, an antibody (or moiety or epitope) that
specif1cally binds to a first target may or may not specifically or preferentially
bind to a second target. As such, “immunospecific binding” does not necessarily
require (although it can include) “exclusive” binding. Generally, but not necessarily,
reference to binding means “immunospecif1c” binding.
As used herein, the term “immunologically active” in reference to an epitope
being or “remaining immunologically active” refers to the capability of an antibody
(e.g, an anti-CD3 antibody) to bind to the epitope under different conditions, for
uvxnkunvv, “LLULVVuJ LwaLALVLLw, w VLI/WLALLLL uvxnkunvv, w VMLVVLLJuwaV, w uvzxLLL, u; w
chemotherapeutic compound. Various compounds can be synthesized, for example,
example, after the epitope has been subjected to reducing and ring conditions.
For example, if the antibody is no longer able to bind a denatured epitope, that epitope
is said to have been rendered immunologically inactive.
Different biological fianctions are associated with the anti-CD3 antibodies of
the present ion, and such antibodies may t any or all of the following
attributes, or may lack, one, two, three or more such attributes: an ability to
specifically bind human CD3 as endogenously expressed on the surface of a normal
human T cell; an ability to specifically bind human CD3 as endogenously expressed
on the surface of a human leukemic T cell; an ability to specifically bind non-human
mammal (e.g., cynomolgus monkey) CD3 as endogenously expressed on the surface
of a normal non-human mammal T cell; an y to specifically bind non-human
CD3 as endogenously expressed on the e of a normal non-human T cell; an
ability to specifically bind a man CD3 as endogenously expressed on the
surface of a non-human leukemic T cell; an y to neutralize (2'.e., block or
interfere with binding) the formation of the CD3 complex; an ability to neutralize the
formation of the TCR complex; an ability to te (either antagonistically or
agonistically) signaling by the TCR complex; an ability to bind the Fc receptor; an
ability to competitively inhibit preferential binding of a known D3 antibody to
CD3, including the ability to preferentially bind to the same CD3 epitope to which the
original antibody preferentially binds; an ability to bind to a portion of CD3 that is
exposed on the surface of a liVing cell in vitro or in viva; an ability to bind to a portion
of CD3 that is exposed on the surface of a living cancer cell; an y to deliver a
chemotherapeutic agent into a cancerous T cell; and/or an ability to deliver a
therapeutic agent, toxin or detectable marker into a T cell. As discussed herein,
polypeptides (including antibodies) of the invention may have any one or more of
these characteristics.
As used herein, the term “agent” refers to a biological, pharmaceutical, or
chemical compound. Non-limiting examples include simple or complex organic or
nic le, a peptide, a protein, an oligonucleotide, an antibody, an antibody
derivative, antibody fragment, a vitamin tive, a ydrate, a toxin, or a
chemotherapeutic compound. s compounds can be synthesized, for example,
ULLVLALVULLVLWIJVMI/LU “5V“"l VJ “LLVVU VLALuLLLb u; LLLuLLVVU VLALuLLLb vuw “wt/MULLLALVLL» uv w
common platform, such that the antibody directs the localization of the agent to the
small molecules and oligomers (e. g., oligopeptides and oligonucleotides), and
synthetic organic compounds based on various core structures. In addition, various
natural sources can provide compounds for screening, such as plant or animal
extracts, and the like. Agents that are employed in the methods of this invention can
be randomly selected or rationally selected or designed. As used , an agent is
said to be randomly selected when the agent is chosen without prior eration or
knowledge of the specific amino acid or other chemical moieties involved in the
association of the molecule with its native binding r(s) or known antibodies.
An example of a randomly selected agent is an agent that is identified h the use
and screening of a chemical library or a peptide combinatorial library. As used
herein, an agent is said to be rationally selected or designed when the agent is chosen
on a non-random basis that takes into account the sequence of the target site and /or
its conformation in connection with the agent's action. Agents can be rationally
selected or ally designed by utilizing the peptide sequences that make up the
contact sites of the receptor /ligand and/or CD3/anti-CD3 antibody complex. For
example, a rationally selected peptide agent can be a peptide whose amino acid
ce is identical to an epitope appearing on CD3 as it is exposed on the surface of
a living cell in its native environment. Such an agent will reduce or block the
ation of the anti-CD3 dy with CD3, or the association of CD3 with its
native ligand, as desired, by binding to the anti-CD3 antibody or to the native ligand.
As used herein, the term “labeled,” with regard to an antibody, is intended to
encompass direct labeling of the antibody by coupling (z'.e., physically linking) a
detectable substance, such as a radioactive agent or a hore (e. g. phycoerythrin
(PE) or cein isothiocyanate (also known as fluoroisothiocyanate or FITC)) to
the dy, as well as indirect labeling of the probe or antibody by reactivity with a
detectable substance.
As used herein, the term “association,” with regard to an antibody, includes
covalent and non-covalent attachment or binding of an agent (e. g., chemotherapeutic
agent) to the dy. The antibody can be associated with an agent (e.g.,
chemotherapeutic agent) by direct g or indirect binding via attachment to a
common rm, such that the antibody directs the localization of the agent to the
such as via targeting and /or internalization, delaying the progression of the disease,
cancerous cell to which the antibody binds and wherein the antibody and agent do not
substantially dissociate under physiological conditions such that the agent is not
targeted to the same cancerous cell to which the antibody binds or such that the
s potency is not decreased.
The term gical sample” encompasses a variety of sample types
obtained from an individual and can be used in a stic or monitoring assay. The
definition encompasses saliva, blood and other liquid samples of biological origin,
solid tissue samples such as a biopsy en or tissue cultures or cells d
rom, and the progeny thereof, for example, cells ed from a tissue sample
ted from an individual suspected of having cancer, in preferred embodiments
from ovary, lung, prostate, pancreas, colon, and breast tissue. The definition also
includes samples that have been manipulated in any way after their ement, such
as by ent with reagents, solubilization, or enrichment for certain components,
such as proteins or polynucleotides, or embedding in a semi-solid or solid matrix for
sectioning purposes. The term “biological sample” encompasses a clinical sample,
and also includes cells in e, cell supematants, cell lysates, serum, plasma,
biological fluid, and tissue samples.
The term “host cell” includes an individual cell or cell culture that can be or
has been a ent for vector(s) for incorporation of polynucleotide s. Host
cells include progeny of a single host cell, and the progeny may not necessarily be
completely identical (in morphology or in genomic DNA complement) to the original
parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells
transfected in vivo with a cleotide(s) of this invention.
As used herein, an “effective amount” of a pharmaceutical composition, in
one embodiment, is an amount sufficient to effect beneficial or desired results
including, without limitation, clinical results such as shrinking the size or rate of
growth of a tumor, delaying or attenuating an inflammatory reaction, increasing the
quality of life of those suffering from a disease, decreasing the dose of other
medications required to treat such disease, enhancing the effect of another medication
such as via targeting and /or internalization, delaying the progression of the disease,
denotes a human.
and/ or prolonging survival of individuals. Such effective amount can be
administered in one or more administrations. For purposes of this invention, an
ive amount of drug, compound, or pharmaceutical composition is an amount
sufficient to rate a clinical observable condition.
In some embodiments, an effective amount of a drug, compound, or
pharmaceutical composition may or may not be achieved in conjunction with another
drug, compound, or pharmaceutical composition. Thus, an “effective amount” may
be ered in the context of administering one or more additional agents, and a
single agent may be considered to be given in an effective amount if, in conjunction
with one or more other agents, a desirable result may be or is achieved. While
individual needs vary, determination of optimal ranges of ive amounts of each
component is within the skill of the art. Typical dosage administered to a patient is
typically 0.0001 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the
dosage administered to a patient is between 0.0001 mg/kg and 20 mg/kg, 0.0001
mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1
mg/kg, 0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to
0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to
0.25 mg/kg or 0.01 to 0.10 mg/kg of the patient's body weight. The dosage and
frequency of administration of molecules of the invention may be reduced or altered
by ing uptake and tissue penetration of the molecules of the invention by
modifications such as, for example, tion.
As used herein, a nucleic acid molecule or agent, antibody, composition or
cell, etc, is said to be “isolated” when that nucleic acid molecule, agent, antibody,
composition, or cell, etc. is ntially separated from inant nucleic acid
molecules, antibodies, agents, itions, or cells, etc. naturally t in its
original source.
The term “individual” refers to a vertebrate animal, preferably a mammal.
Mammals include, but are not limited to, humans, farm animals, sport animals, pets,
es, mice and rats. In the most preferred embodiment, the term individual
denotes a human.
obtaining a beneficial or desired result including and preferably a beneficial or desired
The terms “polypeptide,” “oligopeptide, 3, ide” and “protein” are used
interchangeably herein to refer to polymers of amino acids of any length. The
polymer may be linear or branched, it may comprise modified amino acids, and it may
be interrupted by non-amino acids. The terms also encompass an amino acid polymer
that has been modified naturally or by intervention; for example, disulfide bond
formation, glycosylation, lipidation, acetylation, phosphorylation, or any other
manipulation or modification, such as conjugation with a labeling ent. Also
included within the definition are, for example, polypeptides ning one or more
analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well
as other modifications known in the art. It is understood that, because the
ptides of this invention are based upon an antibody, the polypeptides can occur
as single chains or as ated chains.
As used herein, the term antially pure” refers to material that is at least
50% pure (i.e., free from contaminants), more preferably at least 90 % pure, more
preferably at least 95% pure, more preferably at least 98% pure, more preferably at
least 99% pure, and most ably greater than 99% pure.
As used herein, the term “toxin” refers to any substance which effects an
adverse response within a cell. For example, a toxin ed to a ous cell
would have an adverse, sometimes deleterious effect, on the cancerous cell.
Examples of toxins include, but are not limited to, a taxane, a maytansinoid, an
auristatin (e.g., monomethyl auristatin (MMAE), monomethyl auristatin F (MMAF),
auristatin E (AE), etc.) (such as those disclosed in United States s Nos.
,208,020; 5,416,064; 6,333,410; 701; 6,372,738; 6,436,931; 6,441,163;
6,596,757; 7,276,497; 7,585,857; or 7,851,432), a calicheamicin, an anthracycline
(e.g., doxorubicin), a CC-1065 , docetaxel,; cathepsin B or E; ricin, gelonin,
Pseudomonas exotoxin, diphtheria toxin, and RNase; radiolabeled dies (e.g.,
tiuxetan-conjugated or labeled with a toxic radioisotope (for example, 90Y; 1311, 177Lu,
186 188 211 212225
Re, Re, At, B1, B1, Ac, etc.).
As used herein, the terms “treatment” or “treating” denote an approach for
obtaining a beneficial or desired result including and preferably a beneficial or desired
suspension. ing B-cells, or all dissociated spleen cells, can then be fused with
mveloma cells (e.g.. X63- A98.653 and those from the Salk Institute. Cell
clinical result. Such beneficial or d clinical results include, but are not limited
to, one or more of the following: reducing inflammation or an autoimmune se,
reducing the proliferation of (or destroying) cancerous cells or other diseased cells,
reducing metastasis of cancerous cells found in cancers, shrinking the size of the
tumor, decreasing symptoms resulting from the disease, increasing the quality of life
of those suffering from the disease, decreasing the dose of other medications required
to treat the disease, delaying the progression of the disease, and /or prolonging
al of duals.
11. Methods of Making the Antibodies And Polypeptides 0f the
Present Invention
Methods of making onal antibodies are known in the art. One
method which may be employed is the method of Kohler, G. et al. (1975)
“Continuous Cultures d Cells Secreting Antibody 0fPredefined Specificity,”
Nature 256:495-497 or a modification thereof. lly, onal antibodies are
developed in non-human species, such as mice. In general, a mouse or rat is used for
immunization but other animals may also be used. The antibodies are produced by
immunizing mice with an immunogenic amount of cells, cell extracts, or n
preparations that contain human CD3. The immunogen can be, but is not limited to,
primary cells, cultured cell lines, cancerous cells, nucleic acids, or tissue.
In one embodiment, monoclonal antibodies that bind to CD3 are obtained by
using host cells that over-express CD3 as an immunogen. Such cells e, by way
of example and not by limitation, human T cells.
To monitor the antibody response, a small biological sample (e. g., blood)
may be obtained from the animal and tested for antibody titer against the gen.
The spleen and /or several large lymph nodes can be removed and dissociated into
single cells. If d, the spleen cells may be screened (after removal of non-
specifically adherent cells) by applying a cell suspension to a plate or to a well coated
with the antigen. B-cells, expressing membrane-bound immunoglobulin specific for
the antigen, will bind to the plate, and are not rinsed away with the rest of the
suspension. Resulting B-cells, or all dissociated spleen cells, can then be fused with
myeloma cells (e.g., X63- Ag8.653 and those from the Salk Institute, Cell
four general steps to humanize a monoclonal antibody. These are: (l) determining the
Distribution Center, San Diego, CA). Polyethylene glycol (PEG) may be used to fuse
spleen or lymphocytes with myeloma cells to form a hybridoma. The hybridoma is
then cultured in a selective medium (e. g., hypoxanthine, aminopterin, thymidine
medium, otherwise known as “HAT medium”). The resulting hybridomas are then
plated by limiting dilution, and are d for the production of antibodies that bind
specifically to the immunogen, using, for e, FACS (fluorescence activated cell
g) or immunohistochemistry (IHC) screening. The selected onal
antibody-secreting hybridomas are then cultured either in vitro (e. g., in tissue culture
s or hollow fiber reactors), or in vivo (e. g., as ascites in mice).
As another alternative to the cell ‘l technique, Epstein-Barr Virus
(EBV)—immortalized B cells may be used to produce monoclonal antibodies of the
subject ion. The hybridomas are expanded and subcloned, if desired, and
supematants are assayed for anti-immunogen activity by conventional assay
procedures (e. g., FACS, IHC, radioimmunoassay, enzyme immunoassay, fluorescence
immunoassay, etc.).
In another alternative, anti-CD3 monoclonal antibody and any other
equivalent antibodies can be sequenced and produced recombinantly by any means
known in the art (e. g., humanization, use of transgenic mice to produce fully human
dies, phage display technology, etc.). In one embodiment, anti-CD3
monoclonal antibody is sequenced and the polynucleotide sequence is then cloned
into a vector for expression or propagation. The sequence encoding the antibody of
st may be maintained in a vector in a host cell and the host cell can then be
expanded and frozen for fiJture use.
The cleotide ce of anti-CD3 onal antibody and any
other equivalent antibodies may be used for genetic manipulation to generate a
“humanized” antibody, to improve the affinity, or other characteristics of the
antibody. The general principle in humanizing an antibody involves retaining the
basic sequence of the antigen-binding portion of the antibody, while swapping the
man remainder of the antibody with human antibody sequences. There are
four general steps to humanize a monoclonal antibody. These are: (l) determining the
Daugherty et al. (1991) “Polymerase Chain Reaction Facilitates The Cloning, CDR-
Grafting. And Ranid Expression OfA Murine Monoclonal Antibody Directed Against
nucleotide and predicted amino acid ce of the starting antibody light and heavy
variable domains (2) designing the humanized antibody, i. e., deciding which antibody
ork region to use during the humanizing process (3) the actual humanizing
methodologies /techniques and (4) the transfection and expression of the zed
antibody. See, for example, US. s Nos. 4,816,567; 5,807,715; 692; and
6,33 1,415.
A number of “humanized” antibody molecules comprising an antigenbinding
site derived from a non-human immunoglobulin have been described,
including chimeric antibodies having rodent or modified rodent V regions and their
associated complementarity determining regions (CDRs) fiased to human constant
domains (see, for example, Winter et al. (1991) “Man-made Antibodies,” Nature
349:293-299; Lobuglio et al. (1989) “Mouse/Human Chimeric Monoclonal dy
In Man.‘ Kinetics And Immune Response,” Proc. Natl. Acad. Sci. (U.S.A.) 86:4220-
4224 (1989), Shaw et al. (1987) “Characterization Of A Mouse/Human Chimeric
Monoclonal Antibody (I 7-IA) To A Colon Cancer Tumor-Associated Antigen,” J.
Immunol. 138:4534-4538, and Brown et al. (1987) “Tumor-Specific Genetically
Engineered /Human Chimeric Monoclonal Antibody,” Cancer Res. 47:3577-
3583). Other references be rodent CDRs grafted into a human supporting
framework region (FR) prior to fusion with an appropriate human antibody nt
domain (see, for example, Riechmann, L. et al. (1988) “Reshaping Human dies
for Therapy,” Nature 332:323-327; Verhoeyen, M. et al. (1988) “Reshaping Human
Antibodies: Grafting An Antilysozyme Activity,” e 239:1534-1536; and Jones et
al. (1986) “Replacing The Complementarity-Determining Regions In A Human
Antibody With Those From A Mouse,” Nature 321:522-525). Another reference
describes rodent CDRs supported by recombinantly veneered rodent framework
regions. See, for example, European Patent Publication No. 519,596. These
“humanized” molecules are designed to minimize unwanted immunological response
toward rodent anti-human antibody molecules, which limits the duration and
effectiveness of therapeutic applications of those moieties in human recipients. Other
s of zing antibodies that may also be utilized are disclosed by
rty et al. (1991) “Polymerase Chain Reaction Facilitates The Cloning, CDR-
Grafting, And Rapid Expression OfA Murine onal Antibody Directed Against
vv VAL Mu FULJtJthvLuvu "qu; UULLVL tjvuu wLwLLUwaLVLLVI/L LALVuLLvat/LVLLU’ ”mun; WU, LVL v‘annntjnv,
The CD18 Component 0fLeukocyte Integrins,” Nucl. Acids Res. l9:247l-2476 and
in US. Patents Nos. 6,180,377; 6,054,297; 5,997,867; and 5,866,692.
The invention also asses single chain le region fragments
(“scFV”) of antibodies of this invention, such as mu-anti-CD3. Single chain variable
region fragments are made by linking light and/ or heavy chain le regions by
using a short linking peptide. Bird et al. (1988) (“Single-Chain Antigen-Binding
Proteins,” Science 3-426) describes example of linking peptides which bridge
approximately 3.5 nm between the carboxy terminus of one variable region and the
amino terminus of the other variable region. Linkers of other sequences have been
designed and used (Bird et al. (1988) “Single-Chain Antigen-Binding Proteins,”
Science 242:423-426). Linkers can in turn be modified for additional filnctions, such
as attachment of drugs or attachment to solid supports. The single chain variants can
be ed either recombinantly or synthetically. For tic production of scFV,
an automated synthesizer can be used. For recombinant production of scFV, a suitable
plasmid containing polynucleotide that encodes the scFV can be introduced into a
suitable host cell, either eukaryotic, such as yeast, plant, insect or mammalian cells, or
prokaryotic, such as E. coli. Polynucleotides encoding the scFV of interest can be
made by routine manipulations such as ligation of polynucleotides. The resultant scFV
can be isolated using rd protein purification techniques known in the art.
The invention includes ations to anti-CD3 antibodies and their
binding nts. Modification of polypeptides is routine practice in the art and
need not be described in detail herein. Examples of d polypeptides include
polypeptides with conservative substitutions of amino acid residues, one or more
deletions or additions of amino acids which do not significantly deleteriously change
the onal activity, or use of chemical analogs. Amino acid residues which can be
conservatively substituted for one another include but are not limited to:
glycine/alanine; valine/isoleucine/leucine; asparagine/glutamine; aspartic
acid/glutamic acid; serine/threonine; lysine/arginine; and phenylalanine/tryosine.
These polypeptides also include glycosylated and cosylated polypeptides, as
well as ptides with other ranslational modifications, such as, for example,
glycosylation with ent sugars, acetylation, and phosphorylation. Preferably, the
SF. (1991) “Amino Acid tution Matrices From An ation Theoretic
Dnnnnnnnfinn ” T “linl D1n1 910 <4474A< pnwnnflxr Han mnaf orqxronnnr‘ DT OQTTR/f
amino acid substitutions would be conservative, i.e., the substituted amino acid would
possess similar al properties as that of the original amino acid. Such
conservative substitutions are known in the art, and examples have been provided
above. Amino acid modifications can range from changing or modifying one or more
amino acids to complete redesign of a region, such as the variable region. Changes in
the variable region can alter g y and/or specificity. Other methods of
modification include using coupling ques known in the art, including, but not
limited to, tic means, oxidative substitution and chelation. Modifications can
be used, for example, for attachment of labels for immunoassay, such as the
attachment of radioactive moieties for radioimmunoassay. Modified polypeptides are
made using established procedures in the art and can be screened using standard
assays known in the art.
The fact that a single amino acid alteration of a CDR residue can result in
loss of functional binding (Rudikoff, S. etc. (1982) e Amino Acid Substitution
Altering Antigen-Binding Specificity,” Proc. Natl. Acad. Sci. (USA) 79(6): 1979-1983)
provides a means for systematically identifying alternative functional CDR sequences.
In one preferred method for obtaining such variant CDRs, a polynucleotide encoding
the CDR is mutagenized (for example via random mutagenesis or by a site-directed
method (e.g., polymerase mediated amplification with primers that encode the
mutated locus)) to produce a CDR having a substituted amino acid residue. By
comparing the identity of the relevant residue in the al (filnctional) CDR
sequence to the identity of the substituted unctional) variant CDR sequence, the
BLOSUM62.iij substitution score for that substitution can be identified. The
BLOSUM system provides a matrix of amino acid substitutions created by analyzing
a database of sequences for trusted alignments (Eddy, SR. (2004) “Where Did The
BLOSUM62 ent Score Matrix Come From?,” Nature Biotech. 22(8):1035-
1036; Henikoff, J.G. (1992) “Amino acid substitution matrices from protein blocks,”
Proc. Natl. Acad. Sci. (USA) 15-10919; Karlin, S. et al. (1990) “Methods For
Assessing The Statistical Significance 0f Molecular ce Features By Using
General Scoring Schemes,” Proc. Natl. Acad. Sci. (USA) 87:2264-2268; Altschul,
SF. (1991) “Amino Acid Substitution Matrices From An Information Theoretic
Perspective,” J. Mol. Biol. 219, 555-565. Currently, the most advanced BLOSUM
rather than single nucleotides results in a semi-randomized repertoire of amino acid
database is the BLOSUM62 database (BLOSUM62.iij). Table 1 presents the
BLOSUM62.iij substitution scores (the higher the score the more conservative the
substitution and thus the more likely the substitution will not affect function). If an
antigen-binding fragment comprising the ant CDR fails to bind to CD3, then the
BLOSUM62.iij substitution score is deemed to be insufficiently vative, and a
new candidate substitution is selected and produced having a higher substitution
score. Thus, for e, if the original residue was glutamate (E), and the non-
functional substitute residue was histidine (H), then the BLOSUM62.iij tution
score will be 0, and more conservative changes (such as to aspartate, asparagine,
glutamine, or lysine) are preferred.
Table 1
—----EIIIIP
-l 0 -2 -l -l -l -l -2 -l
0 -2 0 -3 -2 +2 -l -3 -2
+1 -3 -3 0 -2 -3 -2
—1 —1 —3 —4 —1 —3 — 3 -l
—3 —3 —1 —1 —3 —1 —2 -3
—2 0 —3 —2 --1 0 — 3 -l
—2 0 —3 —3 --1 —2 -3 -l 0
6 —2 —4 —4 —2 -3 -3 -2 0
—2 +8 -3 —3 —1 -2 - 1 _2 -l
4 2 -3 --l 0 -3
2 4 -2 --2 -3
-2 -l -3 -l 0
2 "5 0 -2
0 -3 0 +6 -4
- -l -2 —4 7
—2 —2 0 —1 —2 — l 4
-2 -2 —1 —1 —1 —1 —2 —1 1
-2 -2 —3 —2 —3 —1 --1 —4 —3 —
-2 -2 -3 -3 +3 +1 -2 +1 - -2
The invention thus contemplates the use of random mutagenesis to identify
improved CDRs. Phage display technology can alternatively be used to se (or
decrease) CDR affinity. This technology, ed to as affinity maturation, employs
mutagenesis or “CDR walking” and re-selection uses the target antigen or an
antigenic fragment thereof to identify antibodies having CDRs that bind with higher
(or lower) affinity to the n when compared with the initial or parental antibody
(See, e.g. Glaser et al. (1992) J. Immunology 149:3903). Mutagenizing entire codons
rather than single nucleotides results in a semi-randomized repertoire of amino acid
mutations. Libraries can be ucted consisting of a pool of variant clones each of
“Affinity maturation of antibodies assisted by in silico modeling,” Proc. Natl. Acad.
Qni {TTQA\ 1n<l9£\-On90,0n’1/‘ 114 o nrnpnwnr‘ nmlanr‘limnnf «“111fome 14101-00 moxr Ian
which differs by a single amino acid alteration in a single CDR and which contain
variants representing each possible amino acid substitution for each CDR residue.
Mutants with increased (or decreased) g affinity for the antigen can be screened
by contacting the immobilized mutants with labeled antigen. Any screening method
known in the art can be used to identify mutant dies with increased or decreased
affinity to the antigen (e.g., ELISA) (See Wu et al. 1998, Proc Natl. Acad Sci. USA
95:6037; Yelton et al., 1995, J. Immunology 155:1994). CDR walking which
randomizes the light chain may be used possible (See Schier et al., 1996, J. Mol. Bio.
263 :55 1).
Methods for accomplishing such affinity maturation are described for
example in: Krause, J.C. et al. (2011) “An Insertion Mutation That Distorts Antibody
Binding Site Architecture Enhances Function OfA Human Antibody,” MBio. 2(1) pii:
-10. doi: 10.1128/mBio.00345-10; Kuan, C.T. et al. (2010) “Afiinity-Matured
Anti-Glycoprotein NMB Recombinant Immunotoxins Targeting Malignant Gliomas
And Melanomas,” Int. J. Cancer 10.1002/ijc.25645; Hackel, B.J. et al. (2010)
“Stability And CDR Composition Biases Enrich Binder Functionality apes,” J.
Mol. Biol. 401(1):84-96; Montgomery, D.L. et al. (2009) “Afiinity Maturation And
Characterization Of A Human Monoclonal Antibody Against HIV-I gp4I,” MAbs
1(5):462-474; Gustchina, E. et al. (2009) “Afiinity Maturation By Targeted
Diversification Of The CDR-H2 Loop Of A Monoclonal Fab d From A
Synthetic Naive Human Antibody Library And Directed Against The Internal Trimeric
Coiled-Coil 0f Gp4I Yields A Set Of Fabs With Improved HIV-I Neutralization
Potency And Breadth,” Virology 393(1):112-119; Finlay, W.J. et al. (2009) “Afiinity
Maturation OfA Humanized Rat Antibody For Anti-RAGE Therapy.‘ hensive
Mutagenesis Reveals A High Level OfMutational Plasticity Both Inside And e
The Complementarity-Determining Regions,” J. Mol. Biol. 388(3):541-558; m,
J. et al. (2009) ving Antibody g y And Specificity For Therapeutic
Development,” Methods Mol. Biol. 525:353-376; , S. et al. (2008) “In Vitro
Afiinity Maturation Of Human GM—CSF Antibodies By Targeted CDR-
Diversification,” Mol. Immunol. 46(1):135-144; and as, R. et al. (2008)
“Affinity maturation of antibodies assisted by in silico modeling,” Proc. Natl. Acad.
Sci. (USA) 105(26):9029-9034. In a preferred embodiment, multi-well plates may be
Lnnvvnnununn9 “LL “LLwLVVu AMULULL L UULLUWLLLU ULLV u; LALULV u; v wLuv “ULALWLLLU IzLLVI/I/
specifically bind to CD3 and another amino acid sequence to which it is not attached
coated with a selected CD3 antibody (e. g., 100 ng/well in carbonate buffer at room
ature for 2 hrs) and subsequently incubated with soluble CD3 added at a
dilution of 1/10 and incubated at room temperature for 16 hrs or diluted to a
concentration of 50 ng/ml in PBS-T-BSA (0.05 ml added to each well and incubated
for at least 2 h at room temperature). The plate is then washed and dilutions of
recombinant antibodies starting at 0.5 ug/ml in PBS-T-BSA are then added and
incubated for 1 hr at room temp. Binding of recombinant antibodies to the captured
antigen is then measured using, for example, an anti-human IgG-HRP conjugate and
TMB substrate. After stopping color development using dilute sulfuric acid, the plate
is read at 450 nM and higher affinity antibodies identified (see, e.g., United States
Patent No. 7,351,803).
The invention includes polypeptides comprising an amino acid sequence of
the dies of this invention. The ptides of this invention can be made by
procedures known in the art. The polypeptides can be produced by proteolytic or
other degradation of the antibodies, by recombinant methods (i.e., single or fusion
polypeptides) as described above or by chemical synthesis. Polypeptides of the
dies, especially shorter polypeptides up to about 50 amino acids, are
conveniently made by chemical sis. Methods of chemical synthesis are known
in the art and are cially available. For example, an anti-CD3 polypeptide
could be produced by an automated polypeptide synthesizer employing the solid
phase method.
The invention also encompasses fusion proteins comprising one or more
fragments or regions from the polypeptides and antibodies of this invention. In one
embodiment, a fusion polypeptide is provided that comprises at least 10 uous
amino acids of variable light chain region and at least 10 amino acids of variable
heavy chain region. In another embodiment, the fusion polypeptide contains a
heterologous immunoglobulin constant region. In another embodiment, the fusion
polypeptide ns a light chain variable region and a heavy chain variable region of
an antibody ed from a publicly-deposited oma. For es of this
invention, an antibody fusion n contains one or more polypeptide s that
specifically bind to CD3 and another amino acid sequence to which it is not attached
1501'dtlng Ule 'dIlIlDOLlleS rnaue II'Ol'Il HOST 'dIlll'Il'dlS, ODI'dlIllIlg Ule gene sequence, and
using the gene sequence to express the antibody recombinantly in host cells (e.g.,
in the native le, for example, a heterologous sequence or a homologous
sequence from r region.
An anti-CD3 polypeptide, and other CD3 agonists, antagonists and
modulators can be created by s known in the art, for example, tically or
recombinantly. One method of producing such molecules involves chemical
synthesis of the polypeptide, followed by treatment under oxidizing conditions
appropriate to obtain the native mation, that is, the correct disulfide bond
linkages. This can be accomplished using methodologies well known to those skilled
in the art (see, e. g., Kelley, R. F. et al. (1990) In: GENETIC ENGINEERING PRINCIPLES
AND METHODS, Setlow, J.K. Ed., Plenum Press, N.Y., vol. 12, pp 1-19; Stewart, J.M
et al. (1984) SOLID PHASE PEPTIDE SYNTHESIS, Pierce Chemical Co., Rockford, IL;
see also United States s Nos. 603; 3,972,859; 3,842,067; and 3,862,925).
Polypeptides of the invention may be conveniently prepared using solid
phase peptide synthesis (Merrifield, B. (1986) “Solid Phase Synthesis,” e
232(4748):341-347; en, RA. (1985) “General Method For The Rapid Solid-
Phase Synthesis 0f Large Numbers 0f Peptides: Specificity 0f Antigen-Antibody
Interaction At The Level OfIndividual Amino Acids,” Proc. Natl. Acad. Sci. (USA)
82(15):5131-5135; Ganesan, A. (2006) “Solid-Phase sis In The -First
y,” Mini Rev. Med. Chem. 6(1):3-10).
In yet another alternative, fully human antibodies may be obtained through
the use of commercially available mice that have been engineered to express specific
human immunoglobulin proteins. Transgenic animals that are designed to produce a
more desirable (e.g., fully human antibodies) or more robust immune response may
also be used for generation of humanized or human antibodies. Examples of such
technology are XENOMOUSETM (Abgenix, Inc., Fremont, CA) and HUMAB-MOUSE®
and TC M (both from Medarex, Inc., Princeton, NJ).
In an alternative, antibodies may be made recombinantly and expressed
using any method known in the art. Antibodies may be made recombinantly by first
isolating the antibodies made from host animals, obtaining the gene sequence, and
using the gene sequence to express the antibody recombinantly in host cells (e.g.,
CHO cells). Another method that may be employed is to express the antibody
ce in plants {e.g., tobacco) or transgenic milk. Suitable methods for expressing
antibodies recombinantly in plants or milk have been sed (see, for example,
Peeters et al. (2001) “Production 0fAntibodies And Antibody Fragments In Plants,”
Vaccine 19:2756; Lonberg, N. et al. (1995) “Human Antibodies From Transgenic
Mice,” Int. Rev. Immunol 13:65-93; and Pollock et al.(l999) “Transgenic Milk As A
Method For The Production 0f Recombinant Antibodies,” J. Immunol Methods
231:147-157). Suitable methods for making derivatives of antibodies, e. g.,
humanized, single chain, etc. are known in the art. In another alternative, antibodies
may be made recombinantly by phage display technology (see, for example, US.
Patent Nos. 332; 5,580,717; 5,733,743; 6,265,150; and Winter, G. et al. (1994)
“Making dies By Phage Display Technology,” Annu. Rev. Immunol. 12.433-
455).
The antibodies or protein of interest may be subjected to sequencing by
Edman degradation, which is well known to those of skill in the art. The peptide
information generated from mass ometry or Edman degradation can be used to
design probes or primers that are used to clone the protein of interest.
An alternative method of cloning the n of interest is by “panning”
using purified CD3 or portions thereof for cells expressing the antibody or protein of
interest. The “panning” ure may be conducted by obtaining a cDNA library
from tissues or cells that express CD3, over-expressing the cDNAs in a second cell
type, and ing the transfected cells of the second cell type for a specific binding
to CD3. Detailed ptions of the s used in cloning mammalian genes
coding for cell surface proteins by ng” can be found in the art (see, for
example, Aruffo, A. et al. (1987) “Molecular Cloning OfA CD28 cDNA By A High-
Efiiciency COS Cell Expression System,” Proc. Natl. Acad. Sci. (USA) 84:8573-
8577 and Stephan, J. et al. (1999) tive Cloning 0f Cell Surface Proteins
Involved In Organ Development: Epithelial Glycoprotein Is Involved In Normal
Epithelial Difi’erentiation,” Endocrinol. 140:5841-5854).
tJLthLLL u; LLLUVLVL’U vaL vv LuVLLuLLLvu.
cDNAs encoding anti-CD3 antibodies, and other CD3 peptide agonists,
nists and modulators can be obtained by reverse transcribing the mRNAs from
a particular cell type according to standard s in the art. Specifically, mRNA
can be isolated using various lytic enzymes or chemical solutions according to
procedures set forth in, for example, MOLECULAR CLONING: A LABORATORY
MANUAL, Third Edition (Sambrook et al. Eds., 2001) Cold Spring Harbor Press, Cold
Spring Harbor, NY) or ted using commercially available nucleic-acid-binding
resins following the accompanying instructions provided by manufacturers (e.g.,
Qiagen, Invitrogen, Promega). The synthesized cDNAs may then be introduced into
an expression vector to produce the dy or protein of interest in cells of a second
type. It is implied that an expression vector must be replicable in the host cells either
as an episome or as an integral part of the chromosomal DNA. Suitable expression
vectors include but are not limited to plasmids, viral vectors, including adenoviruses,
associated viruses, retroviruses, and cosmids.
The vectors containing the polynucleotides of interest can be introduced into
the host cell by any of a number of appropriate means, including electroporation,
transfection employing calcium chloride, rubidium chloride, m phosphate,
DEAE- n, or other substances; microprojectile bombardment; lipofection; and
infection (e.g., where the vector is an infectious agent such as vaccinia virus). The
choice of introducing vectors or polynucleotides will often depend on features of the
host cell.
Any host cells capable of xpressing logous DNAs can be used
for the purpose of isolating the genes ng the antibody, polypeptide or n of
interest. Non-limiting examples of suitable mammalian host cells include but are not
limited to COS, HeLa, and CHO cells. Preferably, the host cells express the cDNAs at
a level of about 5-fold , more preferably 10-fold higher, even more preferably
-fold higher than that of the corresponding endogenous dy or protein of
interest, if present, in the host cells. Screening the host cells for a specific binding to
CD3 is effected by an immunoassay or FACS. A cell over-expressing the antibody or
n of interest can be identified.
III. Methods for Screening Polypeptides and onal Antibodies
Several methods may be used to screen polypeptides and monoclonal
antibodies that bind to CD3. It is tood that “binding” refers to biologically or
immunologically relevant specific binding, and does not refer to non-specific binding
that may occur, for e, when an immunoglobulin is used at a very high
concentration against a ecific target. In one embodiment, monoclonal
antibodies are screened for binding to CD3 using standard screening techniques. In
this manner, anti-CD3 monoclonal antibody was obtained. The preferred hybridomas
of the present invention are those that produce antibodies mAbl and mAb2, or
ic or humanized tives thereof. However, additional monoclonal
antibodies that bind to CD3 may be identified. For this purpose, monoclonal
antibodies are screened for their differential ability to bind to human CD3 as well as a
primate CD3.
Any of several different detection s may be utilized to detect binding
of antibodies to tissue section. Typically, immunohistochemistry involves the binding
of a primary antibody to the tissue and then a secondary antibody ve against the
s from the primary antibody was generated and conjugated to a detectable
marker (e.g., horseradish peroxidase (HRP), or obenzedine (DAB)). One
alternative method that may be used is polyclonal mirror image complementary
antibodies or polyMICATM (polyclonal Mirror Image Complementary Antibodies;
The Binding Site Limited, Birmingham, UK; Mangham, D.C. et al. (1999) “A Novel
Immunohistochemical Detection System Using Mirror Image Complementary
Antibodies (MICA),” athology 35(2):129-33). The PonMICATM technique can
be used to test binding of primary antibodies (e. g., anti-CD3 antibodies) to normal and
cancerous tissue. Several kinds of polyMICATM Detection kits are commercially
available: Product No. HK004.D is a polyMICATM ion kit which uses DAB
gen; Product No. HK004.A is a polyMICATM Detection kit which uses AEC
chromagen. Alternatively, the primary antibody may be directly labeled with the
detectable marker.
DUPPULL LIIL/ unvuuvu LullL/LLUII U1 LUIIULLUIID UL [JIM PGLLLUUIGI \JUJ PVPLIUV “SULLLDL,
antagonist or modulator. These conjugates include CD3 peptide agonist, antagonist or
IV. s of Characterizing Anti-CD3 Antibodies
Any of several s can be used to terize anti-CD3 antibodies.
One method is to identify the epitope to which it binds. Epitope mapping is
commercially available from various sources, for example, Pepscan Systems
(Lelystad, The Netherlands). Epitope mapping can be used to determine the sequence
to which an anti-CD3 antibody binds. The epitope can be a linear epitope, i.e.,
contained in a single stretch of amino acids, or a conformational epitope formed by a
dimensional ction of amino acids that may not necessarily be contained in
a single h.
Peptides of varying s (e.g., preferably at least 4-6 amino acids long)
can be isolated or synthesized {e.g., recombinantly) and used for binding assays with
anti-CD3 antibody. The epitope to which anti-CD3 antibody binds can be ined
in a atic screening by using overlapping peptides derived from the extracellular
sequence and determining binding by anti-CD3 antibody.
Yet r method that can be used to characterize an anti-CD3 antibody is
to use ition assays with other antibodies known to bind to the same antigen,
i.e., CD3 to determine if anti-CD3 antibodies binds to the same epitope as other
antibodies. Examples of cially available antibodies to CD3 may be available
and may be identified using the binding assays taught herein. Competition assays are
well known to those of skill in the art, and such procedures and illustrative data are
detailed fiarther in the Examples. Anti-CD3 antibodies can be fiarther characterized by
the tissues, type of cancer or type of tumor to which they bind.
V. red Compositions of the Present Invention
The present invention encompasses compositions, including pharmaceutical
compositions, comprising anti-CD3 antibodies, polypeptides derived from anti-CD3
antibodies, polynucleotides comprising sequence encoding anti-CD3 antibodies, and
other agents as described herein. The invention fiarther es for ates of any
CD3 peptide agonist, antagonist or modulator, and additional al structures that
support the intended fianction or functions of the particular CD3 peptide agonist,
antagonist or modulator. These conjugates include CD3 peptide agonist, antagonist or
ing by the TCR complex;
(7) an ability to bind the Fc or;
modulator covalently bound to a macromolecule such as any insoluble, solid support
matrix used in the diagnostic, screening or purification procedures discussed herein.
Suitable matrix materials include any nce that is chemically inert, has high
porosity and has large numbers of functional groups capable of forming covalent
linkages with peptide ligands. es of matrix materials and procedures for
preparation of matrix-ligand conjugates are described in Dean et al. (Eds) AFFINITY
CHROMATOGRAPHY: A PRACTICAL APPROACH, IRL Press (1985); Lowe, “An
Introduction to Afi‘mz’ty Chromatography”, in Work et al. (eds) LABORATORY
TECHNIQUES IN MISTRY AND MOLECULAR Y, Vol. 7, Part 11, North-
Holland (1979); Porath et al., “Biospecz'fic Afi‘mz’ty Chromatography”, in Neurath, H.
et al. (eds), THE PROTEINS, 3rd ed., Vol. 1, pp. 95-178 (1975); and Schott, H.
AFFINITY CHROMATOGRAPHY, Macel Dekker, Inc. NY (1984).
Also ed herein are conjugates of CD3 peptide agonist, antagonist or
modulator and any reporter moiety used in the diagnostic procedures discussed herein.
The CD3 peptide agonist, antagonist or modulator agents, polypeptides and ns
of this invention, including anti-CD3 antibodies, are further identified and
characterized by any (one or more) of the following criteria:
(1) an ability to specifically bind human CD3 as endogenously expressed
on the surface of a normal human T cell;
(2) an ability to specifically bind human CD3 as endogenously expressed
on the surface of a human leukemic T cell;
(3) an ability to specifically bind non-human CD3 (e.g., CD3 of
cynomolgus monkey) as endogenously sed on the surface of a
normal non-human T cell;
(4) an ability to specifically bind a non-human CD3 as endogenously
expressed on the surface of a non-human leukemic T cell;
(5) an ability to neutralize (z'.e. , block or interfere with binding) the
formation of the CD3 complex; an ability to neutralize the formation of
the TCR complex;
(6) an ability to modulate (either antagonistically or tically)
ing by the TCR complex;
(7) an y to bind the Fc receptor;
FVLJtJvtjt/Luvu UVLLLIJLLULLLb “LLJ u; wLLvuv LwaLALVLLt/u “AV LuvLLwLLLVu “LL“ VLLwvavaLuvu VJ
anv (one or more) of the criteria described above.
(8) an y to competitively inhibit preferential binding of a known anti-
CD3 antibody to CD3, ing the ability to preferentially bind to the
same CD3 epitope to which the original antibody preferentially binds;
(9) an ability to bind to a portion of CD3 that is exposed on the surface of
a living cell in vitro or in viva; an ability to bind to a n of CD3
that is exposed on the surface of a living cancer cell;
(10) an ability to deliver a chemotherapeutic agent into a cancerous T cell;
and/or
(1 1) an ability to deliver a therapeutic agent, toxin or detectable marker into
a T cell.
A red antibody of the invention will exhibit differential IHC ng of
tumor tissue relative to normal, non-cancerous tissue, and will moreover be capable of
testing in primate (and particularly cynomolgus monkey) models of antibody efficacy.
Preferred antibodies of the present invention will additionally exhibit ble levels
of affinity and antigen specificity. Preferred antibodies of the present invention will
additionally exhibit desirable levels of modulatory activity and cellular
internalization.
In some embodiments, the antibody of the invention is an dy that is
produced by hybridoma mAbl or oma mAb2, which respectively express
murine antibody mAbl and murine antibody mAb2, or progeny thereof The present
invention also encompasses various formulations of antibodies produced by these
hybridomas and lent antibodies or polypeptide fragments (e.g., Fab, Fab',
F(ab')2 Fv, Fc, etc.), chimeric antibodies, single chain (scFv), mutants thereof, fusion
proteins comprising an antibody n, humanized antibodies, and any other
d configuration of any of these or equivalent antibodies that comprises an
n (CD3), recognition site of the required specificity. The invention also
provides human antibodies displaying one or more of the biological characteristics of
an anti-CD3 family member. The equivalent antibodies of the anti-CD3 family
(including humanized antibodies and human antibodies), polypeptide nts, and
polypeptides comprising any of these fragments are identified and characterized by
any (one or more) of the criteria described above.
of the antibody that has any of the following: at least 5 contiguous amino acids of a
AAAAAAAA AL‘LLA A«I,.I«n1 n«4..'1.,\,;l-. n4. 1AA"; 0 AA«L.‘,.“A“" ,\ “"24" n4. 1AA"; ALA“; 1n
Accordingly, the invention provides any of the following (or compositions,
including pharmaceutical compositions, comprising any of the following): (a) an
antibody produced by the host cell or its progeny; (b) a humanized form of such an
antibody; (c) an dy sing one or more of the light chain and /or heavy
chain variable regions of such an antibody; (d) a ic antibody sing
variable regions homologous or derived from variable regions of a heavy chain and a
light chain of such an antibody, and constant regions homologous or derived from
constant regions of a heavy chain and a light chain of a human antibody; (e) an
dy comprising one or more of the light chain and /or heavy chain CDRs (at least
one, two, three, four, five, or six) of such an antibody; (f) an antibody comprising a
heavy and /or a light chain of such an antibody; (g) a human antibody that is
equivalent to such an antibody. A humanized form of the dy may or may not
have CDRs identical to that original antibody, or antibody produced by the host cell
identified above. Determination of CDR regions is well within the skill of the art.
Other embodiments include antibodies that have at least two, three, four, five, or six
CDR(s) that are substantially homologous to at least two, three, four, five or six CDRs
of an antibody produced from a hybridoma deposited as identified herein, or derived
from such an antibody. It is tood that, for purposes of this invention, binding
specificity and/or overall actiVity is generally retained, although the extent of actiVity
may vary compared to an dy produced by a ted hybridoma (may be
greater or lesser). The invention also provides methods of making any of these
antibodies. Methods of making dies are known in the art and are described
herein.
The invention also provides polypeptides comprising an amino acid
sequence of the antibodies of the invention. In some embodiments, the ptide
comprises one or more of the light chain and /or heavy chain variable regions of the
antibody. In some embodiments, the polypeptide comprises one or more of the light
chain and /or heavy chain CDRs of the antibody. In some embodiments, the
polypeptide comprises three CDRs of the light chain and /or heavy chain of the
antibody. In some embodiments, the polypeptide comprises an amino acid sequence
of the antibody that has any of the following: at least 5 contiguous amino acids of a
sequence of the al antibody, at least 8 contiguous amino acids, at least about 10
Cauuaguuau Luuuugauua LCLCCLCCQL ggaaauugag guua
cctactactg ccagcagtgg tcccggaacc cccctacctt cggcggaggc
accaagctgc agatcaccag a
contiguous amino acids, at least about 15 uous amino acids, at least about 20
uous amino acids, at least about 25 contiguous amino acids, at least about 30
contiguous amino acids, wherein at least 3 of the amino acids are from a variable
region of the antibody. In one embodiment, the variable region is from a light chain of
the al antibody. In another embodiment, the variable region is from a heavy
chain of the antibody. In another embodiment, the 5 (or more) contiguous amino acids
are from a complementarity-determining region (CDR) of the antibody.
In some embodiments of this invention, cells of this invention that express
CD3, a portion of CD3, anti-CD3 antibodies or other CD3-binding polypeptides of
this invention are administered directly to an individual to modulate in viva CD3
biological activity.
] The preferred anti-CD3 antibodies of the present invention are mAbl and
mAb2, and humanized or chimeric derivatives and antigen-binding fragments f
that are reactive toward the human and cynomolgus CD3 molecule. The amino acid
and encoding polynucleotide sequences of the le light chain and variable heavy
chain of murine antibodies mAbl and mAb2 are shown below. The sequences of the
CDRs of the exemplary antibodies (mAbl and mAb2) are shown in boldface and
underlined.
A. Sequences of Variable Regions of Murine Monoclonal
Antibody mAbl
Amino Acid Sequence of Murine Monoclonal Antibody mAbl Variable
Light Chain (SEQ ID NO:1):
QVVLTQSPA" MSAEPGLKVT MTCSASSSVS QKSG IYE§
SKLASGVPAR FSGSGSGTSY SET SSMflTfl DAATYYCQQW SRNPPTFGGG
TKLQITR
Polynucleotide ce Encoding VIurine Monoclonal Antibody mAbl
Variable Light Chain (SEQ ID NO:2):
caggeggegc Lgacccagtc ccccgccaec gcce eccccggcga
gaaagtgaca atgacctgc: ccgcceccec chc eacaLgaacL
ggeaecagca gaagtccggc acctccccca agcggeggae cLacgachc
tccaagc:gg cctccggcg: gcccgccaga Lececeggce ccggctccgg
ceac Lcccegacca echceccaL ggaaaccgag gacgccgcca
cc:actactg ccagcagtgg :cccggaacc cccctacct: cggcggaggc
accaagc:gc agatcaccag a
gaggtgaagc egc eggaaag cggcggagga ctgg:gcagc caaaggga":C
achaaach ecc egcgccg cctccggctt cacc:ttaac gc ":6.
Amino Acid Sequence of Murine Monoclonal Antibody mAbl Variable
Heavy Chain (SEQ ID NO:3):
SGA'_‘'.L4 LARPGASVKM SCKASGYTFT RSTMHWVKQa PGQGT.«.W G:
INPSSAYTNY NQKFKDKATL TADKSSSTAY MQLSSLTS'.L'.4 3 SAVYYCASP_Q
VHYDYNGFPY VTVS S
] Polynucleotide Sequence Encoding mAbl Murine Monoclonal Antibody
Variable Heavy Chain (SEQ ID NO:4):
caggtgcagc :gcagcag:c tggcgccgag ctggccagac ctggcgcctc
chgaagaeg echgcaagg cc:ccggcta caccttcacc acca
ggg: gaaacagcgg cc:ggacagg gcctggaatg gatcggc:ac
a:caacccc: ccagcgcc:a caccaac:ac aaccagaagt tcaaggacaa
ggccaccc:g accgccgaca agtcctccag caccgchac aegcagcege
cctccc:gac ctccgaggac tccgccgtgt actactgcgc thcccccag
gegcaceacg actacaacgg cLecccceac ngggccagg gcaccctgg:
gacagtgtcc tcc
B. Sequences of Variable Regions of Murine Monoclonal
Antibody mAbZ
Amino Acid Sequence Of Murine Monoclonal Antibody mAb2 Variable
Light Chain (SEQ ID NO:5):
QAVVTQESAL TTSPGETVTL TCRSSTGAVT WVQE KPDHLFTGL:
GGTNKRAPGV PARFSGSL:G DKAALTITGA QTflDflA YhC ALWYSNLWVF
GGGTKLTVLG
Polynucleotide ce Encoding Murine Monoclonal Antibody n1Ab2
le Light Chain (SEQ ID NO:6):
caggccgtgg agga chagceceg accacatccc caggcgaaac
agegacheg achgcagaL ctgg agcagegace achceaacL
acchaaeeg gngcaggag aagcccgacc accegeecac egggcegaec
ggcggaacca acaaaagggc acccggegeg chgcccgge eeLchgcag
Lcegaecgga gacaaggccg chLgacaae Laceggcgcc gagg
aLgaagceaL LLacLLchL gcachegg aLagcaaLce gagggegLeL
gggggtggca ccaaactgac agtgctggga
Amino Acid Sequence of mAb2 Murine Monoclonal Antibody Variable
Heavy Chain (SEQ ID NO:7):
SGGG LVQPKGSLK; SCAASGFTFN TYAMNWVRQA WVAR
IRSKYNNYAT YYADSVKDRF TISRDDSQS" LYLQMNNHKT EDTAMYYCVR
HGNFGNSYVS WFAYWGQGT; VTVSA
Polynucleotide Sequence Encoding Murine Monoclonal Antibody n1Ab2
Variable Heavy Chain (SEQ ID NO:8):
aagc egc eggaaag cggcggagga ctgg:gcagc caaaggga":C
achaaach ecc g cctccggctt cacc:ttaac acatacgc ":6.
tgaattgggt gcgacaggca cctggcaagg gccsggagsg aagg
tcca agtacaacaa saLgcaacc sacsasgccg ac:ctgtgaa
ggasagaLsc agtc attc ccagagcass csgsaschc
agatgaacaa scsgaaaac: accg ccasgsacsa Lugsgsgcgg
cacggtaact :cggcaassc sachgscL gcss assggggaca
ggggacacsg ngachsgs c chc
Position 40 of the heavy chain is a high affinity MHC class II binding
peptide anchor residue. Positions 44, 48, 54, 94, 99 and108 of the heavy chain are
moderate y MHC class II binding peptide anchor residues. Position 69 of the
light chain is a high affinity MHC class II g peptide anchor residue. Position 59
of the light chain is a moderate affinity MHC class II binding peptide anchor residue.
These es may be substituted, using standard molecular biology techniques, to a
residue in order to reduce or remove the MHC class II recognition site.
C. Fc-Engineered CD3 Antibodies
In traditional immune function, the interaction of antibody-antigen
complexes with cells of the immune system results in a wide array of responses,
ranging from effector functions such as antibody-dependent cytotoxicity, mast cell
degranulation, and phagocytosis to modulatory signals such as regulating
lymphocyte proliferation and antibody secretion. All of these ctions are initiated
through the binding of the Fc domain of antibodies or immune complexes to
specialized cell surface receptors on hematopoietic cells. The diversity of cellular
responses triggered by antibodies and immune complexes results from the structural
heterogeneity of the three Fc receptors: FcyRI (CD64), FcyRII (CD32), and FcyRIII
. FcyRI (CD64), FcyRIIA (CD32A) and FcyRIII (CD16) are activating (z'.e.,
immune system enhancing) receptors; FcyRIIB (CD32B) is an inhibiting (z'.e.,
immune system dampening) receptor. The amino acid sequence of the IgGl Fc
region is shown below (as SEQ ID NO:9, numbered according to Kabat et al.,
SEQUENCE OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service,
NIH, MD (1991), expressly incorporated herein by reference, and hereafter referred to
as “Kabat EU”). Residues 230-341 are the Fc CH2 region. Residues 342-447 are the
Fc CH3 region:
lVVAA/J LLVLVLWVLJ’ I/LLV VLLLuLLLb FLUtJVLIILVU u; uLLv LALuvavau u; uLLv Lnnvvnnununn “AV
SEQIDNO:9
PAPELLGGPS VFLFPPKPKD THM SRTPflV TCVVVDVSifl DPflVKhNWYV
230 240 250 260 270
DGVEVHNA<T KPRflflQYNST YRVVSVLTVL HQDWLNGKEY KC<VSNKALP
280 290 300 310 320
AP flKT S<A KGQPREPQVY THPPSRflflMT KNQVSLTCLV KGFYPSDIAV
330 340 350 360 370
flWflSNGQPflN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH
380 390 400 410 420
?AHHNHYTQK SLSLSPGK
430 440
Since Fc receptor (FcR)-non-binding CD3-specific dies are minimally
ing, it has been proposed that they may alter TCR s in a way that might
induce immune tolerance (St. Clair E.W. (2009) “Novel Targeted Therapies for
Autoimmunity,” Curr. Opin. Immunol. 21(6):648-657). Thus, such therapy has
potential application in the treatment of mune disease and host vs. graft tissue
rejection. FcR non-binding CD3-specific antibodies have also been postulated to
induce remission in type 1 diabetes mellitus tolerance (St. Clair E.W. (2009) “Novel
Targeted Therapies for Autoimmunity,” Curr. Opin. Immunol. 21(6):648-657;
Masharani, U.B. et al. (2010) “Teplizumab Therapy For Type 1 Diabetes,” Expert
Opin. Biol. Ther. 10(3):459-465).
] The present invention thus es antibodies that specifically bind to CD3
that comprise a variant Fc region haVing Fc regions that are modified (e.g.,
substitutions, deletions, insertions in one or more portions) so as to be unable or less
able to bind to the Fc or (relative to an antibody haVing the same CDRs but a
wild-type Fc region).
In one embodiment, such antibodies will be incapable of binding to any PC
receptor. Alternatively, the Fc region of the antibody will be modified so as to permit
it to bind to Fc receptors such as B that are inhibitory, but not to Fc receptors
such as A, FcyRIIIA or IB that promote activation of the immune
system.
Preferably, the binding properties of the molecules of the invention are
characterized by in vitro filnctional assays for determining one or more FcyR mediator
1\11UVV11 LU U11V D1\111\/u 111 L11\/ “1L cuLu VAV111P1111VU 11V1V111. 111V nupp “DD“.YD UDVU 111
effector cell functions. The affinities and binding properties of the les, e.g.,
antibodies, of the ion for an FcyR can be determined using in vitro assays
(biochemical or immunological based assays) known in the art for determining
antibody-antigen or Fc-FcyR interactions, z'.e., specific binding of an antigen to an
antibody or specific binding of an Fc region to an FcyR, respectively, including but
not limited to ELISA assay, surface plasmon resonance assay, immunoprecipitation
assays. In most preferred embodiments, the molecules of the invention have similar
binding properties in in vivo models (such as those described and disclosed herein) as
those in in vitro based assays. However, the present invention does not exclude
molecules of the invention that do not exhibit the desired phenotype in in vitro based
assays but do exhibit the desired phenotype in viva.
In some embodiments, the les of the invention comprising a variant
Fc region comprise at least one amino acid modification (for example, possessing l,
2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) in the CH3 domain of the Fc
region, which is defined as ing from amino acids 342-447. In other
embodiments, the molecules of the invention comprising a variant Fc region comprise
at least one amino acid ation (for example, possessing l, 2, 3, 4, 5, 6, 7, 8, 9,
or more amino acid modifications) in the CH2 domain of the Fc region, which is
defined as extending from amino acids 231-341. In some embodiments, the
molecules of the invention comprise at least two amino acid modifications (for
example, possessing 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications), wherein
at least one such modification is in the CH3 region and at least one such ation
is in the CH2 region. The invention filrther encompasses amino acid modification in
the hinge region. In a particular ment, the invention encompasses amino acid
modification in the CH1 domain of the Fc region, which is defined as extending from
amino acids 2 1 6-230.
In particularly preferred embodiments, the ion asses molecules
comprising a variant Fc region wherein said t confers or has a decreased ADCC
ty and/or a decreased binding to A (CD32A), as measured using s
known to one skilled in the art and exemplified herein. The ADCC assays used in
Antibodies With Increased Activity To Recruit Complement,” J. Immunol. 1662571-
accordance with the methods of the invention may be NK dependent or macrophage
dependent.
In particularly preferred embodiments, the invention encompasses les
comprising a variant Fc region n said variant confers or has a decreased ADCC
activity and/or a decreased binding to IA (CDl6A), as measured using methods
known to one skilled in the art and exemplified herein. The ADCC assays used in
accordance with the methods of the invention may be NK dependent or macrophage
dependent.
The PC variants of the present invention may be ed with other PC
modifications, such as those disclosed in United States Patents Nos. 7,632,497;
7,521,542; 619; 7,416,727; 7,371,826; 7,355,008; 742; 7,332,581;
7,183,387; 7,122,637; and 6,737,056; in PCT Publications Nos. ;
; ; ; WO 06/088494; WO
05/115452; WO 05/110474; WO 04/1032269; and in WO 04/063351; and in Presta,
L.G. et al. (2002) “Engineering therapeutic antibodies for improved function,”
Biochem. Soc. Trans. 30(4):487-490; Shields, R.L. et al. (2002) “Lack offucose on
human IgG1 N-linked oligosaccharide improves binding to human chamma RIII and
antibody-dependent cellular toxicity,” J. Biol. Chem. 26;277(30):26733-26740 and
Shields, R.L. et al. (2001) “High resolution mapping of the binding site on human
IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1
variants with improved binding to the Fc gamma R,” J. Biol. Chem. 276(9):659l-
6604). The invention encompasses combining an Fc variant of the ion with
other PC ations to e ve, synergistic, or novel properties to the
modified antibody.
In other embodiments, the ion encompasses the use of any PC variant
known in the art, such as those disclosed in Jefferis, B.J. et al. (2002) action
Sites 0n Human IgG-Fc For chammaR.‘ Current Models,” Immunol. Lett. 82:57-65;
Presta, L.G. et al. (2002) “Engineering Therapeutic Antibodies For Improved
Function,” Biochem. Soc. Trans. 30:487-90; Idusogie, E.E. et al. (2001) “Engineered
Antibodies With Increased Activity To Recruit Complement,” J. Immunol. 1662571-
75; Shields, R.L. et al. (2001) “High Resolution Mapping Of The Binding Site On
Human IgGI For Fc Gamma RI, Fc Gamma RII, Fc Gamma RIII, And FcRn And
Design Of IgGI Variants With Improved Binding To The Fc gamma R,” J. Biol.
Chem. 276:6591-6604; Idusogic, E.E. et al. (2000) “Mapping Of The CIq Binding
Site On Rituxan, A Chimeric Antibody With A Human IgG Fc,” J. Immunol.
164:4178-84; Roddy, M.P. et al. (2000) nation 0f Fc Receptor-Dependent
r Functions Of A d IgG4 Monoclonal Antibody To Human CD4,” J.
Immunol. 164:1925-1933; Xu, D. et al. (2000) “In Vitro Characterization of Five
Humanized OKT3 Eflector Function Variant dies,” Cell. Immunol. 200:16-26;
Armour, K.L. et al. (1999) “Recombinant human IgG Molecules Lacking chamma
Receptor I Binding And Monocyte Triggering Activities,” Eur. J. Immunol. -
24; Jeffcris, R. et al. (1996) “Modulation 0ch(Gamma)R And Human ment
Activation By IgG3-Core Oligosaccharide Interactions,” Immunol. Lett. 54:101-04;
Lund, J. et al. (1996) “Multiple Interactions OfIgG With Its Core Oligosaccharide
Can Modulate Recognition By Complement And Human Fc Gamma Receptor I And
Influence The Synthesis OfIts Oligosaccharide Chains,” J. Immunol. 157:4963-4969;
Hutchins et al. (1995) “Improved tribution, Tumor Targeting, And Reduced
Immunogenicity In Mice With A Gamma 4 Variant 0f Campath-IH,” Proc. Natl.
Acad. Sci. (USA) 92:11980-84; chfcris, R. et al. (1995) “Recognition Sites 0n
Human IgG For Fc Gamma ors.‘ The Role Of Glycosylation,” Immunol. Lett.
44:111-17; Lund, J. et al. (1995) saccharide-Protein Interactions In IgG Can
Modulate Recognition By Fc Gamma ors,” FASEB J. 9:115-19; Alcgrc, ML.
et al. (1994) “A Non-Activating "Humanized"Anti-CD3 Monoclonal Antibody Retains
Immunosuppressive ties In Vivo,” Transplantation 57:1537-1543; Lund et al.
(1992) ple Binding Sites On The CH2 Domain OfIgG For Mouse Fc Gamma
RII,” Mol. Immunol. 29:53-59; Lund et al. (1991) “Human Fc Gamma RI And Fc
Gamma RII Interact With Distinct But Overlapping Sites 0n Human IgG,” J.
Immunol. 147:2657-2662; Duncan, A.R. et al. (1988) “Localization Of The Binding
Site For The Human High-Afiinity Fc Receptor 0n IgG,” Nature 332:563-564; US
Patent Nos. 5,624,821; 5,885,573; 6,194,551; 7,276,586; and 7,317,091; and PCT
Publications WO 00/42072 and PCT WO 99/5 8572.
In certain ments, the antibody of the invention comprises a variant Fc
region (including an Fc derived from any human immunoglobulin type (e.g., IgG,
IgE, IgM, IgD, IgA and IgY), or class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) or
subclass), wherein said variant Fc region comprises at least one amino acid
modification relative to a wild-type Fc region, which variant Fc region exhibits
d or hed binding to one or more effector ligands as determined by
standard assays known in the art and disclosed herein, relative to a able
molecule comprising the wild type Fc region. In certain embodiments, the variant Fc
domain of the antibody of the invention comprises an amino acid modification (i.e.,
insertion, substitution, deletion) at one or more of the residues 233, 234, 235, 236,
237, 238, 265, 270, 297, 298, 299. In a specific embodiment, the one or more amino
acid modifications which reduce or abolish binding to one or more effector ligands is
a substitution with phenylalanine or proline at position 233; a substitution with
alanine at position 234; a substitution with alanine or ic acid at position 235; a
substitution with alanine at on 236, a substitution with alanine at position 237, a
substitution with arginine at on 238; a substitution with alanine or glutamic acid
at position 265; a substitution with alanine or asparagine at position 270; a
tution with alanine or glutamine at position 297; a substitution with
phenylalanine, asparagine or e at position 298; a substitution with any amino
acid at position 299 other than serine or threonine; or a combination of two or more of
the above-listed substitutions. In certain embodiments, the antibody of the invention
comprises an Fc domain having a substitution with alanine at position 265 and at
position 297; a tution with alanine at position 265 and with ine at
position 297; a substitution with ic acid at position 265 and with alanine at
position 297; or a substitution with glutamic acid at position 265 and with glutamine
at position 297. In preferred embodiments, the antibody of the invention comprises
an Fc domain having a modification (e.g., substitution, insertion, deletion) at on
234 and position 235 of the Fc region. In a specific example in accordance with this
embodiment, the antibody of the invention comprises an Fc domain having a
substitution at position 234 with e and a substitution at position 235 with
glutamic acid. In a yet more preferred embodiment, the antibody of the invention
comprises an Fc having a substitution with alanine at position 234 and a substitution
with alanine at position 235.
In other embodiments, the antibody of the invention comprises a Fc region,
which t Fc region exhibits reduced or abolished binding to one or more effector
ligands as determined by standard assays known in the art and disclosed herein,
relative to a comparable control molecule. In certain embodiments, the antibody of
the invention has a Fc region that exhibits reduced or abolished binding to one or
more effector s, which Fc region comprises a phenylalanine or proline at
position 233; an alanine at position 234; an alanine or glutamic acid at position 235;
an alanine at position 236, an alanine at position 237, an arginine at position 238; an
alanine or glutamic acid at position 265; an alanine or asparagine at position 270; an
alanine or glutamine at on 297; a alanine, asparagine or proline at
position 298; any amino acid at position 299 other than serine or threonine; or a
ation of two or more of the above-listed substitutions. In certain
embodiments, the antibody of the ion comprises an Fc domain having an
alanine at position 265 and at position 297; an alanine at position 265 and a glutamine
at position 297; a glutamic acid at position 265 and an alanine at position 297; or a
glutamic acid at position 265 and a glutamine at position 297. In certain
embodiments, the antibody of the ion comprises an Fc domain having an
alanine at 234 and a glutamic acid at position 235. In preferred embodiments, the
antibody of the invention comprises an Fc having an alanine at position 234 and an
alanine at position 235.
dies of the invention that comprise and Fe domain having an alanine
at positions ponding to 234 and 235 according to the ing scheme of
Kabat are known as “ala-ala” dies. In certain embodiments, use of “ala-ala” Fc
domains and/or other combinations of amino acid combinations herein bed
ding combinations comprising “ala-ala” Fc domains) may abolish binding of the
Fc domain to all FcyRs. The binding of a PC domain to one or more FcyRs may be
determined by any method described herein and/or known in the art.
LAvauvu vu VJ LAvauvuLLLb UVVLvuLuLL u; VJ vaxLLLvu LLLULMuLLLb up.» LLVI/ LLLLLLUV“ uv ALLUVLLVML‘LLL
-2 (IL-2). Interleukin-4 (IL-4), Interleukin-6 (IL-6), Interleukin-l2 (IL-12),
In certain embodiments, the one or more amino acid modifications which
abolish binding to all FcyRs or reduce or abrogate binding to one or more or
ligands comprise combinations of the modifications listed herein or combinations of
the modifications listed herein with any that may confer null binding to any FcR (e.g.
FcyRIIIA, FcyRIIIB, FcyRIIA) as determined by the methods disclosed herein or
known to one skilled in the art. As readily understood by one of skill in the art, such
antibodies of the invention may find particular use in the treatment of an autoimmune
disease in that the anti-CD3 antibodies and antigen-binding fragments serve to
te immune filnction without the associated first-dose response common to anti-
immune cell antibodies.
In certain embodiments, the anti-CD3 dies and antigen-binding
fragments of the invention, or antigen binding fragments f, have diminished
(such as, but not limited to, less than 50%, less than 40%, less than 30%, less than
%, less than 10%, less than 5% or less than 1% as compared to binding by a protein
sing a l Fc domain) or, more preferably, no detectable binding to one or
more of any FcyR (e.g., FcyRI, FcyRII or I) via its Fc domain as determined by
assays routine in the art. In addition or alternatively, the anti-CD3 antibodies and
antigen-binding fragments of the ion, or antigen binding fragments thereof, may
have diminished (such as, but not limited to, less than 50%, less than 40%, less than
%, less than 20%, less than 10%, less than 5% or less than 1% as ed to
binding by a control protein sing a l Fc domain) or, more preferably, no
detectable binding to any complement receptors, such as, Clq, as determined in
routinely used assays. In particular embodiments, the antibody is aglycosylated. In
other embodiments, the antibody lacks an Fc domain (e.g., is a Fab fragment, F(ab’)2
or single chain antibody).
The antibodies of the invention are thus particularly useful because they have
reduced or no in viva ty caused by lymphokine production or ne release.
Methods of measuring lymphokine production and cytokine release are known and
routine in the art and encompassed . For example, cytokine release may be
measured by measuring secretion of cytokines including but not limited to Interleukin
-2 (IL-2). Interleukin-4 (IL-4), Interleukin-6 (IL-6), Interleukin-l2 (IL-12),
domains (A) and (E) associate to form a binding site that binds epitope (1); said
Interleukin-16 (IL-16), PDGF, TGF-OL, TGF-B, TNF- 0t, TNF- B, GCSF, GM-CSF,
MCSF, IFN— 0t, IFN— [3, TEN-y, IGF-I, IGF-II. For example, see, Isaacs et al., 2001,
Rheumatology, 40: 8; Soubrane et a1., 1993, Blood, 81(1): 15-19; each of
which is incorporated herein by reference in its entirety.
D. CD3 DARTTM Diabodies
As discussed above, the present invention additionally encompasses
ific, trispecific and mutispecific antibodies. A ularly preferred example of
such antibodies comprise M” diabody molecules that comprise at least two
polypeptide chains which form at least two epitope binding sites, at least one of which
specifically binds to CD3. Exemplary M” diabody molecules are disclosed in
US20100174053, US20090060910, US20070004909, EP2158221, EP1868650,
WO2010080538, WO2008157379, and WO2006113665.
In preferred embodiments, the first polypeptide chain of the DARTTM
diabody ses:
(i) a domain (A) comprising a binding region of a light chain variable
domain of a first immunoglobulin (VLl) specific for an epitope (1);
(ii) a domain (B) comprising a binding region of a heavy chain variable
domain of a second immunoglobulin (VH2) specific for an epitope (2);
(iii) a domain (C).
The second polypeptide chain of such a DARTTM diabody comprises:
(i) a domain (D) comprising a binding region of a light chain le
domain of the second immunoglobulin (VL2) c for epitope (2);
(ii) a domain (E) comprising a binding region of a heavy chain variable
domain of the first immunoglobulin (VH1) specific for epitope (1); and
(iii) a domain (F).
The DARTTM diabody domains (A) and (B) do not ate with one another to form
an epitope g site. Similarly, the DARTTM diabody domains (D) and (E) do not
associate with one another to form an epitope binding site. Rather, DARTTM diabody
domains (A) and (E) associate to form a binding site that binds epitope (1); said
any affinity based method known in the art or exemplified herein, e.g., affinity
tography.
DARTTM diabody domains (B) and (D) associate to form a binding site that binds said
epitope (2). Domains (C) and (F) are covalently associated together.
Each polypeptide chain of the DARTTM diabody molecule comprises a VL
domain and a VH domain, which are covalently linked such that the domains are
constrained from self-assembly. Interaction of two of the polypeptide chains will
produce two VL-VH pairings, forming two eptipoe binding sites, z'.e., a bivalent
molecule. Neither the VH or VL domain is constrained to any position within the
ptide chain, l'.e., restricted to the amino (N) or carboxy (C) teminus, nor are the
domains restricted in their relative ons to one another, z'.e., the VL domain may
be inal to the VH domain and vice-versa. The only restriction is that a
complimentary ptide chain be ble in order to form functional DARTTM
diabodies. Where the VL and VH domains are derived from the same antibody, the
two complimentary polypeptide chains may be identical. For example, where the
binding domains are derived from an antibody specific for epitope A (i.e., the binding
domain is formed from a A interaction), each polypeptide will comprise a
VHA and a VLA. Homodimerization of two polypeptide chains of the dy will
result in the formation two VLA-VHA binding sites, resulting in a bivalent
monospecific antibody. Where the VL and VH domains are derived from antibodies
specific for different antigens, formation of a onal bispecific DARTTM diabody
requires the interaction of two different polypeptide chains, z'.e., formation of a
heterodimer. For example, for a bispecific DARTTM diabody, one ptide chain
will comprise a VLA and a VLB; homodimerization of said chain will result in the
formation of two VLA-VHB binding sites, either of no binding or of unpredictable
binding. In st, where two differing polypeptide chains are free to interact, e.g.,
in a inant expression system, one comprising a VLA and a VHB and the other
comprising a VLB and a VHA, two differing binding sites will form: VLA-VHA and
VLB-VHB. For all DARTTM diabody polypeptide chain pairs, the possibly of
misalignment or mis-binding of the two chains is a possibility, z'.e., interaction of VL-
VL or VH-VH domains; however, purification of filnctional diabodies is easily
managed based on the immunospecificity of the ly dimerized binding site using
any affinity based method known in the art or exemplified , e.g., affinity
chromatography.
to form a monomer, and said monomers interact via their ed Fc domains to
One or more of the polypeptide chains of the DARTTM diabody may
optionally comprise an Fc domain domain or portion thereof (6.g. a CH2 domain, or
CH3 domain). The PC domain or n thereof may be d from any
immunoglobulin e or allotype including, but not limited to, IgA, IgD, IgG, IgE
and IgM. In preferred embodiments, the Fc domain (or portion thereof) is derived
from IgG. In specific embodiments, the IgG isotype is IgGl, IgG2, IgG3 or IgG4 or
an allotype thereof. In one embodiment, the diabody molecule comprises an Fc
domain, which Fc domain comprises a CH2 domain and CH3 domain ndently
selected from any immunoglobulin isotype (i.e. an Fc domain comprising the CH2
domain derived from IgG and the CH3 domain d from IgE, or the CH2 domain
derived from IgGl and the CH3 domain derived from IgG2, etc.). The PC domain
may be engineered into a polypeptide chain sing the diabody molecule of the
invention in any position relative to other domains or portions of said polypeptide
chain (e.g., the Fc domain, or portion thereof, may be c-terminal to both the VL and
VH domains of the polypeptide of the chain; may be n-terminal to both the VL and
VH domains; or may be N-terminal to one domain and c-terminal to another (i.e.,
between two domains of the ptide chain)).
The PC domains in the polypeptide chains of the DARTTM diabody molecules
preferentially dimerize, resulting in the formation of a DARTTM molecule that
exhibits immunoglobulin-like properties, e.g., Fc-FcyR, interactions. Fc comprising
diabodies may be dimers, e. g., comprised of two polypeptide , each comprising
a VH domain, a VL domain and an Fc domain. Dimerization of said polypeptide
chains results in a bivalent DARTTM diabody comprising an Fc domain, albeit with a
structure distinct from that of an unmodified bivalent antibody. Such DARTTM
diabody molecules will t altered phenotypes relative to a ype
immunoglobulin, e.g., altered serum half-life, binding properties, etc. In other
embodiments, DARTTM diabody les comprising Fc domains may be tetramers.
Such tetramers comprise two ‘heavier’ polypeptide chains, z'.e., a polypeptide chain
comprising a VL, a VH and an Fc domain, and two ‘lighter’ polypeptide chains, z'.e.,
polypeptide chain comprising a VL and a VH. The lighter and r chains interact
to form a monomer, and said monomers ct via their unpaired Fc domains to
cancer, bladder cancer, kidney cancer, brain , gleoblastoma, thyroid cancer,
form an Ig-like molecule. Such an Ig-like DARTTM diabody is alent and may be
monospecific, bispecific or tetraspecific.
VI. Therapeutic s of Using the Anti-CD3 Antibodies of the
Present invention
The anti-CD3 antibodies of the present invention and their antigen-binding
fragments have particular utility in the treatment of cancers ated With CD3
sion and in the treatment of autoimmune disease and other inflammatory
disorders.
These uses can involve the formation of a complex between CD3 and an
antibody that binds specifically to CD3. Examples of such antibodies include but are
not limited to anti-CD3 monoclonal dies mAbl and mAb2 or, more preferably,
their humanized derivatives. The formation of such a complex can be in vitro or in
viva. Without being bound by theory, anti-CD3 antibody can bind to CD3 through the
extracellular domain of CD3 and may then be internalized inside of a living normal or
cancer cell.
A. Treatment of Cancer
The antibodies and antigen-binding fragments of the present invention bind
to CD3 present on the surface of T cells. The antigen-binding nts of the
present invention can be used in the context of a bi-speciflc (or trispecific or
multispecific) molecule, such as a DART or BiTE molecule, to redirect T-cells to a
tumor cell. The T-cell can then kill the tumor cell. The bispecific (or trispecific or
multispecific) molecules of the t invention are capable of binding to both
human CD3 and the CD3 of a non-human mammal (e.g., cynomolgus monkey), and
also to a second (or additional) and different antigen(s) or epitope(s). The second
antigen or epitope is preferably a tumor antigen expressed on a tumor cell. Such
tumor cells may be from s, for example, breast cancer, prostate , gastric
cancer, lung , stomach cancer, colon cancer, rectal cancer, pancreatic cancer,
liver cancer, ovarian cancer, oral cavity cancer, pharyngeal cancer, esophageal cancer,
laryngeal cancer, bone cancer, skin cancer, melanoma, uterine cancer, testicular
, bladder cancer, kidney cancer, brain cancer, gleoblastoma, thyroid cancer,
The antibodies and antigen-binding nts of the present invention bind
to CD3 present on the surface of T cells. Using conventional methods, such
lymphoma, a, and leukemia. Such The additional antigens or epitopes are
preferably cell surface tumor antigens or epitopes (such as: l7-lA, A33, adult
erythrocyte primary rm I antigen, alpha otein, an envelope antigen of an
RNA tumor Virus, bladder tumor oncofetal n, B7-Hl, B7-H2, B7-H3, B7-H4,
B7-H5, B7-H6, Burkitt's lymphoma n-38.13, CAl25, CD18, CD19, human B-
lymphoma antigen-CD20, CD22, CD33, CD44, CD52, CEA, COl7-lA, CTA-l,
CTLA-4, epidermal grth factor or, Ep-CAM, EphA2, fetal ocyte I
antigen, fibrosarcoma antigen, ganglioside GD2, ganglioside GD3, ganglioside GM2,
ganglioside GM3, GICA 19-9, gp IIIb/IIIa, gp72, HERl, HER-2/neu, HER3, HER4,
high molecular weight melanoma antigen, HLA-DR antigen, human leukemia T cell
antigen-Gp37, human lung carcinoma antigen L20, human lung carcinoma antigen
L6, human milk fat globule antigen, IgE, KS l/4 pan-carcinoma antigen, LEA, lung
adenocarcinoma F3 antigen, ant human lymphocyte antigen-APO-l, melanoma
antigen gp75, melanoma-associated antigen p97, coprotein, nuC242,
polymorphic epithelial mucin antigen, prostate ic antigen, prostate specific
membrane antigen, prostatic acid phosphate, SK-l antigen, TAG-72, T-antigen, tumor
antigen CAl25, tumor antigen MUCl, tumor-specific transplantation type of cell-
surface n, vascular endothelial growth factor, vascular endothelial grth
factor-receptor, and aVB3). Alternatively, such additional antigens or epitopes may be
associated with a pathogen (such as: hepatitis type A, hepatitis type B, hepatitis type
C, influenza, varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplex
type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial
Virus, papilloma Virus, papova Virus, cytomegalovirus, echinovirus, arbovirus,
huntaVirus, coxsackie Virus, mumps Virus, measles Virus, a Virus, polio Virus,
small pox, Epstein Barr Virus, human immunodeficiency Virus type I (HIV-I), human
immunodeficiency Virus type II (HIV-II), Viral miningitis, Viral encephalitis, dengue,
small pox; cteria rickettsia, asma, Neisserz'a, S. pneumonia, Borrelia
burgdorferi, Bacillus anthracis, Streptococcus, Staphylococcus, Mycobacterz'um,
tetanus, pertissus, cholera, , diptheria, chlamydia, and legionella; leishmania,
kokzidioa, trypanosoma or malaria; chlamydia and rickettsia.
The antibodies and antigen-binding fragments of the present invention bind
to CD3 present on the surface of T cells. Using conventional methods, such
the present invention could be employed in the same way as the CD3 antibodies of
antibodies may be labeled with fluorescein, as described above. When such labeled
molecules are incubated in the presence of a if1c molecule (such as for example,
a UDARTTM diabody having an epitope g domain that binds to the T-cell
receptor and an epitope binding domain that binds to fluorescein (“TCRUDARTTM”
)), they can bind to the fluorescein label and thereby localize themselves
to the surface of cells that express CD3 and cause redirected killing of such cells.
In an alternative embodiment, CD19 may be used as the “second” e,
such that a bispecif1c antibody, or more preferably, a DART TM diabody, recognizing
CD3 and CD19 is employed to eradicate B-cell lymphoma through co-engagement of
the B-cell specific antigen (CD19) and the T-cell or/CD3 complex on effector
T-cells. As disclosed by Moore, RA. et al. (2011) “Application Of Dual Afiinity
Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing 0f B-Cell
Lymphoma,” Blood 2011 blood09-306449, a CD3/CD19 DARTTM diabody was
used to eradicate B-cell lymphoma through co-engagement of the B-cell specific
antigen CD19 and the T-cell receptor/CD3 complex on effector s. Side by side
comparison with a single-chain bispecif1c antibody bearing identical CD19 and CD3
antibody Fv sequences revealed the DART to be more potent in directing B-cell lysis.
The enhanced activity with the CDl9xCD3 DART was observed on all CD19
expressing B-cell target cells evaluated using resting and pre-stimulated human
PBMC or purified or T-cell populations. Characterization of a CDl9xTCR
bispecif1c DART revealed equivalent potency to the CDl9xCD3 DART
demonstrating flexibility of the DART architecture to t T-cell/B-cell
associations for redirected T-cell killing applications. Importantly the enhanced level
of g mediated by DART molecules was unaccompanied with any increase in
non-specif1c T-cell activation or lysis of CD19 negative cells. Cell association studies
indicate the DART ecture is well suited for maintaining cell:cell contact
apparently contributing to the high level of target cell killing. y, the ability of
the CDl9xTCR DART to inhibit B-cell ma in NOD/SCID mice when co-
administered with human PBMC r trates the value of DART molecules
for the treatment of B-cell malignancies. The cross-reactive anti-CD3 dies of
the present invention could be employed in the same way as the CD3 antibodies of
and/or reduce the need for other therapy (e.g., 1n the treatment or prophylax1s of Type
I Aiokofon flan mofhnr‘n AP {-140 invronfinn movr 1110 {-140 Moor] ‘an‘ Avnn-onn110 1.1401111.“
Moore, PA. et al. Thus, the invention provides a therapy for cancers (especially
lymphomas and leukemias) involving CD3-expressing cancer cells.
The bispecific (or trispecific or multispecific) les of the present
invention are preferably administered to a t in one or more unit doses of
typically 0.0001 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the
dosage administered to a patient is between 0.0001 mg/kg and 20 mg/kg, 0.0001
mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1
mg/kg, 0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to
0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to
0.25 mg/kg or 0.01 to 0.10 mg/kg of the patient's body weight.
B. Treatment of Autoimmune Disease and Inflammation
The invention also provides methods of treating, preventing, slowing the
progression of and/or ameliorating the symptoms of T-cell ed diseases or
disorders, including graft rejection, graft versus host disease, unwanted delayed-type
hypersensitivity reactions (such as delayed-type allergic reactions), T-cell ed
pulmonary diseases, and autoimmune diseases. T-cell mediated pulmonary diseases
include sarcoidosis, hypersensitivity pneumonitis, acute interstitial nitis,
alveolitis, pulmonary fibrosis, idiopathic pulmonary is and other diseases
characterized by inflammatory lung damage. T-cell autoimmune diseases e
multiple sclerosis, is, polymyositis, sis, vitiligo, Sjogren's syndrome,
rheumatoid arthritis, Type 1 diabetes, autoimmune pancreatitis, inflammatory bowel
diseases (e.g., Crohn's disease and ulcerative colitis), celiac e,
glomerulonephritis, scleroderma, dosis, autoimmune thyroid diseases (e. g.,
Hashimoto's thyroiditis and Graves’ disease), myasthenia , Addison's disease,
autoimmune uveoretinitis, pemphigus vulgaris, primary biliary cirrhosis, pernicious
anemia, and systemic lupus erythematosis, lupus (particularly, cutaneous), effects
from organ lantation, graft vs. host disease (GVHD), etc. Particularly, the
methods of the invention are advantageous in subjects with early stage disease to slow
or reduce the damage from the autoimmunity and maintain a high level of fianction
and/or reduce the need for other therapy (e.g., in the treatment or prophylaxis of Type
I diabetes, the methods of the invention may reduce the need for exogenous insulin
ug/kg or less, 175 ug/kg or less, 150 ug/kg or less, 128 ug/kg or less, 100 ug/kg or
administration in the subject). In addition, the methods of the invention may
advantageously reduce the incidence of or result in no incidence of cytokine release
syndrome previously associated with administration of therapeutic antibodies, and, in
particular, anti-T-cell (e.g., anti-CD3 antibody or antigen-binding fragments.
In certain embodiments, the course of ent with an anti-CD3 antibody
or antigen-binding fragments according to the methods of the invention is repeated at
2 month, 4 month, 6 month, 8 month, 9 month, 10 month, 12 month, 15 month, 18
month, 24 month, 30 month, or 36 month intervals. In specific embodiments efficacy
of the treatment with an anti-CD3 antibody or antigen-binding nts of the
invention is ined as described herein or as is known in the art at 2 months, 4
months, 6 months, 9 months, 12 months, 15 months, 18 months, 24 months, 30
months, or 36 months subsequent to the previous treatment.
In r embodiment, a subject is administered one or more unit doses of
approximately 05-50 ug/kg, approximately 05-40 ug/kg , approximately 05-30
ug/kg, approximately 05-20 ug/kg, approximately 05-15 ug/kg, approximately 0.5-
ug/kg, approximately 0.5-5 ug/kg, approximately 1-5 ug/kg, approximately 1-10
ug/kg, approximately 20-40 ug/kg, imately 20-30 ug/kg, approximately 22-28
ug/kg or approximately 25-26 ug/kg of one or more anti-CD3 antibody or nbinding
fragments to prevent, treat or ameliorate one or more symptoms of an
autoimmune er or T cell malignancy. In another embodiment, a subject is
administered one or more unit doses of 200 ug/kg, 178 ug/kg, 180 ug/kg, 128 ug/kg,
100 ug/kg, 95 ug/kg, 90 ug/kg, 85 ug/kg, 80 ug/kg, 75 ug/kg, 70 ug/kg, 65 ug/kg, 60
ug/kg, 55 ug/kg, 50 ug/kg, 45 ug/kg, 40 ug/kg, 35 ug/kg, 30 ug/kg, 26 ug/kg, 25
ug/kg, 20 ug/kg, 15 ug/kg, 13 ug/kg, 10 ug/kg, 6.5 ug/kg, 5 ug/kg, 3.2 ug/kg, 3
Mg/kg,2.5 , 2 lug/kg, 1.6 lug/kg, 1.5 lug/kg, 1 lug/kg, 0.5 lug/kg, 0.25 lug/kg, 0.1
ug/kg, or 0.05 ug/kg of one or more D3 antibody or antigen-binding fragments
to prevent, treat or ameliorate one or more symptoms of an autoimmune disorder or
T-cell ancy.
] In a one embodiment, a subject is administered one or more doses of 200
ug/kg or less, 175 ug/kg or less, 150 ug/kg or less, 128 ug/kg or less, 100 ug/kg or
u; w va VAL vv VVAx “LL“ LLVI/ wuLALLLLLut/VLLLLb uuuvu u; ULLV tJLthLLJ WLLJ u; I/LLVLWIJVMULUWLLJ
effective amount of one or more anti-CD3 antibody or antigen-binding fragments on
less, 95 ug/kg or less, 90 ug/kg or less, 85 ug/kg or less, 80 ug/kg or less, 75 ug/kg or
less, 70 ug/kg or less, 65 ug/kg or less, 60 ug/kg or less, 55 ug/kg or less, 50 ug/kg or
less, 45 ug/kg or less, 40 ug/kg or less, 35 ug/kg or less, 30 ug/kg or less, 25 ug/kg or
less, 20 ug/kg or less, 15 ug/kg or less, 10 ug/kg or less, 5 ug/kg or less, 2.5 ug/kg or
less, 2 ug/kg or less, 15 ug/kg or less, 1 ug/kg or less, 0.5 ug/kg or less, 0.25 ug/kg
or less, 0.1 ug/kg or less, or 0.05 ug/kg or less of one or more anti-CD3 antibody or
antigen-binding fragments of the invention to prevent, treat or ameliorate one or more
symptoms of an autoimmune disorder or T-cell malignancy.
In particular embodiments, a subject is administered one or more doses of
about 5 - l200 ug/mz, preferably, 51 - 826 ug/mz. In another embodiment, a subject
is administered one or more unit doses of 1200 ug/mz, ll50 ug/mz, 1100 ug/mz, 1050
ug/mz, 1000 ug/mz, 950 Mg/l’nz, 900 Mg/l’nz, 850 Mg/l’nz, 800 ug/m2, 750 Mg/l’nz, 700
Mg/l’nz, 650 Mg/l’nz, 600 Mg/l’nz, 550 z, 500 Mg/l’nz, 450 Mg/l’nz, 400 Mg/l’nz, 350
Mg/l’nz, 300 Mg/l’nz, 250 ug/m2, 200 Mg/l’nz, 150 Mg/l’nz, 100 Mg/l’nz, 50 Mg/l’nz, 40 Mg/l’nz,
ug/m2, 20 ug/mz, l5 ug/mz, 10 ug/mz, or 5 ug/m2 of one or more anti-CD3
antibody or antigen-binding fragments to prevent, treat, slow the progression of, delay
the onset of or ameliorate one or more ms of an autoimmune disorder or T-cell
malignancy.
In another ment, the subject is administered a treatment regimen
comprising one or more doses of a prophylactically or eutically effective
amount of one or more anti-CD3 antibody or antigen-binding fragments, wherein the
course of ent is administered over 2 days, 3 days, 4 days, 5 days, 6 days, 7 days,
8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days. In one embodiment,
the treatment regimen comprises administering doses of the prophylactically or
therapeutically effective amount of one or more anti-CD3 dy or antigen-binding
fragments every day, every 211d day, every 3rd day or every 4th day. In n
ments, the treatment regimen comprises administering doses of the
prophylactically or therapeutically ive amount of one or more anti-CD3
antibody or antigen-binding fragments on Monday, Tuesday, Wednesday, Thursday
of a given week and not administering doses of the prophylactically or therapeutically
effective amount of one or more anti-CD3 dy or antigen-binding fragments on
minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 10 minutes,
Friday, Saturday, and Sunday of the same week until 14 doses, 13, doses, 13 doses, 12
doses, 11 doses, 10 doses, 9 doses, or 8 doses have been administered. In certain
embodiments the dose stered is the same each day of the regimen. In certain
embodiments, a subject is administered a treatment regimen comprising one or more
doses of a prophylactically or therapeutically effective amount of one or more anti-
CD3 antibody or antigen-binding fragments, wherein the prophylactically or
eutically effective amount is 200 ug/kg/day, 175 ug/kg/day, 150 ug/kg/day, 125
ug/kg/day, 100 ug/kg/day, 95 ug/kg/day, 90 ug/kg/day, 85 ug/kg/day, 80 ug/kg/day,
75 ug/kg/day, 70 ug/kg/day, 65 ug/kg/day, 60 ug/kg/day, 55 ug/kg/day, 50
ug/kg/day, 45 ug/kg/day, 40 ug/kg/day, 35 day, 30 ug/kg/day, 26 ug/kg/day,
ug/kg/day, 20 ug/kg/day, 15 ug/kg/day, 13 ug/kg/day, 10 ug/kg/day, 6.5
ug/kg/day, 5 day, 3.2 ug/kg/day, 3 ug/kg/day, 2.5 ug/kg/day, 2 ug/kg/day, 1.6
ug/kg/day, 1.5 ug/kg/day, 1 ug/kg/day, 0.5 ug/kg/day, 0.25 day, 0.1 day,
or 0.05 day; and/or Wherein the prophylactically or therapeutically effective
amount is 1200 ug/mZ/day, 1150 ug/mZ/day, 1100 ug/mZ/day, 1050 ug/mZ/day, 1000
ug/mZ/day, 950 day, 900 ug/mZ/day, 850 ug/mZ/day, 800 ug/mZ/day, 750
ug/mZ/day, 700 ug/mZ/day, 650 ug/mZ/day, 600 ug/mZ/day, 550 ug/mZ/day, 500
ug/mZ/day, 450 day, 400 ug/mZ/day, 350 ug/mZ/day, 300 ug/mZ/day, 250
ug/mZ/day, 200 ug/mZ/day, 150 ug/mZ/day, 100 ug/mZ/day, 50 ug/mZ/day, 40
ug/mZ/day, 30 ug/mZ/day, 20 ug/mZ/day, 15 day, 10 ug/mZ/day, or 5 ug/mZ/day.
In another embodiment, the intravenous dose of 1200 ug/m2 or less, 1150 ug/m2 or
less, 1100 ug/m2 or less, 1050 ug/m2 or less, 1000 ug/m2 or less, 950 ug/m2 or less,
900 ug/m2 or less, 850 ug/m2 or less, 800 ug/m2 or less, 750 ug/m2 or less, 700 ug/m2
or less, 650 ug/m2 or less, 600 ug/m2 or less, 550 ug/m2 or less, 500 ug/m2 or less, 450
ug/m2 or less, 400 ug/m2 or less, 350 ug/m2 or less, 300 ug/m2 or less, 250 ug/m2 or
less, 200 ug/m2 or less, 150 ug/m2 or less, 100 ug/m2 or less, 50 ug/m2 or less, 40
ug/m2 or less, 30 ug/m2 or less, 20 ug/m2 or less, 15 ug/m2 or less, 10 ug/m2 or less, or
ug/m2 or less of one or more anti-CD3 antibody or antigen-binding fragments is
administered over about 24 hours, about 22 hours, about 20 hours, about 18 hours,
about 16 hours, about 14 hours, about 12 hours, about 10 hours, about 8 hours, about
6 hours, about 4 hours, about 2 hours, about 1.5 hours, about 1 hour, about 50
minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 10 minutes,
of 5 until the daily prophylactically or therapeutically effective amount of one or more
anLr‘nq nnfil’xnr‘lv nr nnfimfinJfiinrlinn fc ic anhipvpr‘l
about 5 s, about 2 minutes, about 1 minute, about 30 seconds or about 10
seconds to prevent, treat or ameliorate one or more ms of an mune
disease or T-cell malignancy. The total dosage over the duration of the regimen is
preferably a total of less than 9000 ug/mz, 8000 ug/mz, 7000 ug/mz, 6000 ug/mz, and
may be less than 5000 ug/l’nz, 4000 ug/l’nz, 3000 ug/mz, 2000 ug/m2,01‘1000 ug/l’nz.
In specific embodiments, the total dosage administered in the n is 100 ug/m2 to
200 Mg/l’nz, 100 11ng to 500 ug/mz, 100 Mg/l’nz to 1000 Mg/l’nz, or 500 Mg/l’nz to 1000
ug/mz.
In preferred embodiments, the dose escalates over the first fourth, first half
or first 2/3 ofthe doses (e.g., over the first 2, 3, 4, 5, or 6 days ofa 10, 12, 14, 16, 18
or 20 day regimen of one dose per day) of the treatment regimen until the daily
prophylactically or therapeutically ive amount of one or more anti-CD3
antibody or antigen-binding fragments is achieved. In certain embodiments, a subject
is administered a treatment regimen comprising one or more doses of a
prophylactically or therapeutically effective amount of one or more anti-CD3
antibody or antigen-binding nts, wherein the prophylactically or
therapeutically effective amount is increased by, e. g., 0.01 ug/kg, 0.02 ug/kg, 0.04
ug/kg, 0.05 ug/kg, 0.06 ug/kg, 0.08 ug/kg, 0.1 ug/kg, 0.2 ug/kg, 0.25 ug/kg, 0.5
rig/kg, 0-75 rig/kg, 1 , 1-5 lug/kg 2 rig/kg, 4 rig/kg, 5 rig/kg, 10 Mil/kg, 15 ug/kg,
ug/kg, 25 ug/kg, 30 ug/kg, 35 ug/kg, 40 ug/kg, 45 ug/kg, 50 ug/kg, 55 ug/kg, 60
ug/kg, 65 ug/kg, 70 ug/kg, 75 ug/kg, 80 ug/kg, 85 ug/kg, 90 ug/kg, 95 ug/kg, 100
ug/kg, or 125 ug/kg each day; or increased by, e.g., l ug/mz, 5 ug/mz, 10 ug/mz, l5
ug/l’nz, 20 ug/l’nz, 30 ug/mz, 40 ug/l’nz, 50 ug/l’nz, 60 ug/mz, 70 ug/mz, 80 z, 90
ug/mz, 100 ug/mz, 150 Mg/l’nz, 200 Mg/l’nz, 250 Mg/l’nz, 300 Mg/l’nz, 350 ug/mz, 400
ug/mz, 450 ug/mz, 500 ug/mz, 550 ug/mz, 600 ug/mz, or 650 ug/mz, each day as
treatment progresses. In certain embodiments, a subject is administered a treatment
regimen comprising one or more doses of a prophylactically or therapeutically
effective amount of one or more anti-CD3 dy or antigen-binding fragments,
wherein the prophylactically or therapeutically ive amount is increased by a
factor of 1.25, a factor of 1.5, a factor of 2, a factor of 2.25, a factor of 2.5, or a factor
of 5 until the daily prophylactically or therapeutically effective amount of one or more
anti-CD3 antibody or antigen-binding fragments is achieved.
uuuv u; Avv rub/1x5 u; Lvuu, 1.1 rub/1x5 u; Lvuu, 1v rub/1x5 u; Lvuu, Uu rub/1x5 u; Lvuu, uv
ug/kg or less, 75 ug/kg or less, 70 ug/kg or less, 65 ug/kg or less, 60 ug/kg or less, 55
In a specific embodiment, a t is intramuscularly administered one or
more doses of a 200 ug/kg or less, ably 175 ug/kg or less, 150 ug/kg or less,
125 ug/kg or less, 100 ug/kg or less, 95 ug/kg or less, 90 ug/kg or less, 85 ug/kg or
less, 80 ug/kg or less, 75 ug/kg or less, 70 ug/kg or less, 65 ug/kg or less, 60 ug/kg or
less, 55 ug/kg or less, 50 ug/kg or less, 45 ug/kg or less, 40 ug/kg or less, 35 ug/kg or
less, 30 ug/kg or less, 25 ug/kg or less, 20 ug/kg or less, 15 ug/kg or less, 10 ug/kg or
less, 5 ug/kg or less, 2.5 ug/kg or less, 2 ug/kg or less, 1.5 ug/kg or less, 1 ug/kg or
less, 0.5 ug/kg or less, or 0.5 ug/kg or less of one or more anti-CD3 antibody or
antigen-binding nts to prevent, treat or ameliorate one or more symptoms of an
autoimmune disorder or T-cell malignancy.
In another embodiment, a subject is subcutaneously administered one or
more doses of a 200 ug/kg or less, preferably 175 ug/kg or less, 150 ug/kg or less,
125 ug/kg or less, 100 ug/kg or less, 95 ug/kg or less, 90 ug/kg or less, 85 ug/kg or
less, 80 ug/kg or less, 75 ug/kg or less, 70 ug/kg or less, 65 ug/kg or less, 60 ug/kg or
less, 55 ug/kg or less, 50 ug/kg or less, 45 ug/kg or less, 40 ug/kg or less, 35 ug/kg or
less, 30 ug/kg or less, 25 ug/kg or less, 20 ug/kg or less, 15 ug/kg or less, 10 ug/kg or
less, 5 ug/kg or less, 2.5 ug/kg or less, 2 ug/kg or less, 1.5 ug/kg or less, 1 ug/kg or
less, 0.5 ug/kg or less, or 0.5 ug/kg or less of one or more anti-CD3 antibody or
antigen-binding nts to prevent, treat or ameliorate one or more symptoms of an
autoimmune disorder.
In another embodiment, a subject is intravenously administered one or more
doses ofa 100 ug/kg or less, preferably 95 ug/kg or less, 90 ug/kg or less, 85 ug/kg or
less, 80 ug/kg or less, 75 ug/kg or less, 70 ug/kg or less, 65 ug/kg or less, 60 ug/kg or
less, 55 ug/kg or less, 50 ug/kg or less, 45 ug/kg or less, 40 ug/kg or less, 35 ug/kg or
less, 30 ug/kg or less, 25 ug/kg or less, 20 ug/kg or less, 15 ug/kg or less, 10 ug/kg or
less, 5 ug/kg or less, 2.5 ug/kg or less, 2 ug/kg or less, 1.5 ug/kg or less, 1 ug/kg or
less, 0.5 ug/kg or less, or 0.5 ug/kg or less of one or more D3 antibody or
antigen-binding fragments to prevent, treat or ameliorate one or more symptoms of an
autoimmune disorder or T-cell malignancy. In another embodiment, the intravenous
dose of 100 ug/kg or less, 95 ug/kg or less, 90 ug/kg or less, 85 ug/kg or less, 80
ug/kg or less, 75 ug/kg or less, 70 ug/kg or less, 65 ug/kg or less, 60 ug/kg or less, 55
ug/kg or less, 50 ug/kg or less, 45 ug/kg or less, 40 ug/kg or less, 35 ug/kg or less, 30
ug/kg or less, 25 ug/kg or less, 20 ug/kg or less, 15 ug/kg or less, 10 ug/kg or less, 5
ug/kg or less, 2.5 ug/kg or less, 2 ug/kg or less, 15 ug/kg or less, 1 ug/kg or less, 0.5
ug/kg or less, or 0.5 ug/kg or less of one or more anti-CD3 antibody or antigenbinding
fragments is administered over about 6 hours, about 4 hours, about 2 hours,
about 1.5 hours, about 1 hour, about 50 minutes, about 40 s, about 30 minutes,
about 20 minutes, about 10 minutes, about 5 minutes, about 2 minutes, about 1
minute, about 30 seconds or about 10 seconds to prevent, treat or ameliorate one or
more symptoms of an autoimmune disorder or T-cell ancy.
In specific embodiments in which ting doses are administered for the
first days of the dosing regimen, the dose on day l of the regimen is 5 - 100
ug/mZ/day, and tes to the daily dose as recited immediately above by day 3, 4, 5,
6 or 7. For example, on day l, the t is administered a dose of approximately 51
ug/mZ/day, on day 2 approximately 103 ug/mZ/day, on day 3 approximately 207
ug/mZ/day, on day 4 approximately 413 ug/mZ/day and on subsequent days of the
regimen (e.g., days 5-14) 826 ug/mZ/day.
In other embodiments, the initial dose is 1/4, to 1/2, to equal to the daily dose
at the end of the regimen but is administered in portions at als of 6, 8, 10 on 12
hours. For example, a 13 ug/kg/day dose is administered in four doses of 3-4 ug/kg at
intervals of 6 hours to reduce the level of cytokine release caused by stration of
the antibody.
In c embodiments, to reduce the possibility of cytokine release and
other adverse effects, the first 1, 2, 3, or 4 doses or all the doses in the regimen are
administered more slowly by intravenous administration. For example, a dose of 51
ug/mZ/day may be administered over about 5 minutes, about 15 minutes, about 30
minutes, about 45 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours,
about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about
18 hours, about 20 hours, and about 22 hours. In certain embodiments, the dose is
administered by slow infiJsion over a period of, e.g., 20 to 24 hours. In specific
VALLuLLLb LwaLALVLLt/u ALL ULLV UMVJVUI/ “LI/VA wuLALLLLLUwwaLVLL. ALL yv; UWLLL VLALVUuLLALVLLt/U, ULLV
level of free anti-CD3 antibody or antigen-binding fragments should not exceed 200
embodiments, the dose is infused in a pump, preferably increasing the concentration
of dy administered as the 111filS1011 progresses.
In other embodiments, a set on of the n may be administered in
escalating doses. For example, for the 51 ug/mz/day to 826 ug/mZ/day regimen
described above, the fraction may be 1/10, 1/4, 1/3, 1/2, 2/3 or 3/4 ofthe daily doses.
ingly, when the fraction is 1/ 10, the daily doses will be 5.1 ug/m2 on day 1,
.3 ug/m2 on day 2, 20.7 ug/m2 on day 3, 41.3 ug/m2 on day 4 and 82.6 ug/m2 on
days 5 to 14. When the fraction is 1/4, the doses will be 12.75 ug/m2 on day 1, 25.5
ug/m2 on day 2, 51 ug/m2 on day 3, 103 ug/m2 on day 4, and 207 ug/m2 on days 5 to
14. When the fraction is 1/3, the doses will be 17 ug/m2 on day 1, 34.3 ug/m2 on day
2, 69 ug/m2 on day 3, 137.6 ug/m2 on day 4, and 275.3 ug/m2 on days 5 to 14. When
the fraction is 1/2, the doses will be 25.5 ug/m2 on day l, 51 ug/m2 on day 2, 103
ug/m2 on day 3, 207 ug/m2 on day 4, and 413 ug/m2 on days 5 to 14. When the
fraction is 2/3, the doses will be 34 ug/m2 on day 1, 69 ug/m2 on day 2, 137.6 ug/m2
on day 3, 275.3 ug/m2 on day 4, and 550.1 ug/m2 on days 5 tol4. When the fraction is
3/4, the doses will be 38.3 ug/m2 on day 1, 77.3ug/m2 on day 2, 155.3 ug/m2 on day 3,
309.8 ug/m2 on day 4, and 620 ug/m2 on days 5 to 14.
In specific embodiments, the anti-CD3 antibody or antigen-binding
fragments is not stered by daily doses over a number of days, but is rather
administered by infusion in an uninterrupted manner over 4 hours, 6 hours, 8 hours,
hours, 12 hours, 15 hours, 18 hours, 20 hours, 24 hours, 30 hours or 36 hours. The
111filS1011 may be constant or may start out at a lower dosage for, for example, the first
1, 2, 3, 5, 6, or 8 hours of the infusion and then increase to a higher dosage thereafter.
Over the course of the infusion, the patient receives a dose equal to the amount
administered in the exemplary regimens set forth above. For e, a dose of
approximately 150 ug/mz, 200 ug/mz, 250 ug/mz, 500 ug/mz, 750 ug/mz, 1000 ug/mz,
1500 Mg/l’nz, 2000 Mg/l’nz, 3000 Mg/l’nz, 4000 Mg/l’nz, 5000 Mg/l’nz, 6000 Mg/l’nz, 7000
ug/mz, 8000 ug/mz, or 9000 ug/mz. In particular, the speed and on of the
on is designed to minimize the level of free anti-CD3 antibody or antigenbinding
fragments in the subject after administration. In certain embodiments, the
level of free anti-CD3 antibody or antigen-binding fragments should not exceed 200
amount of one or more anti-CD3 antibody or n-binding fragments does not
ng/ml free antibody. In on, the infusion is ed to achieve a ed T
cell receptor g and modulation of at least 50%, 60%, 70%, 80%, 90%, 95% or
of 100%.
] In certain embodiments, the anti-CD3 antibody or antigen-binding fragments
is administered so as to achieve a certain level of combined g and modulation of
T cell receptor complexes on T cells, as determined by methods well known in the art,
see, e. g., Example ll of US. patent application publication US 2003/0108548, which
is hereby incorporated by reference in its entirety. In ic embodiments, the
dosing regimen achieves a combined T cell receptor coating and modulation of at
least 50%, 60%, 70%, 80%, 90%, 95% or of 100% with, in c embodiments,
little to no free anti-CD3 antibody or antigen-binding fragments detected (for
example, less than 200 ng/mL of the drug is detected in the blood of the patient).
In preferred embodiments, the anti-CD3 antibody or antigen-binding
fragments are administered parenterally, for example, intravenously, intramuscularly
or subcutaneously, or, alternatively, are administered orally. The anti-CD3 antibody
or antigen-binding fragments may also be administered as a sustained e
formulation.
In a specific embodiment, the administration of one or more doses or a
dosage regimen of a prophylactically or therapeutically effective amount of one or
more anti-CD3 antibody or antigen-binding fragments does not induce or reduces
relative to other immunosuppressive agents one or more of the following unwanted or
adverse effects: vital sign abnormalities (fever, tachycardia, bardycardia,
hypertension, nsion), hematological events (anemia, lymphopenia, leukopenia,
thrombocytopenia), headache, chills, dizziness, nausea, asthenia, back pain, chest pain
(chest pressure), diarrhea, myalgia, pain, pruritus, psoriasis, rhinitis, sweating,
injection site reaction, latation, an increased risk of opportunistic ion,
tion of Epstein Barr Virus, apoptosis of T cells and an increased risk of
developing certain types of cancer. In another specific embodiment, the
administration of one or more doses of a prophylactically or therapeutically effective
amount of one or more anti-CD3 antibody or antigen-binding fragments does not
antibody or antigen-binding fragments may be administered to a subject when, for
example, the subject's HA 1 or HA 1 C levels at 1 month, 2 , 4 months, 6
induce or reduces relative to other immunosuppressive agents one or more of the
ing unwanted or adverse effects: vital sign abnormalities (fever, tachycardia,
bardycardia, hypertension, hypotension), hematological events (anemia, lymphopenia,
leukopenia, ocytopenia), headache, chills, dizziness, nausea, asthenia, back
pain, chest pain (chest pressure), diarrhea, myalgia, pain, pruritus, psoriasis, is,
sweating, injection site reaction, vasodilatation, an sed risk of opportunistic
infection, Epstein Barr Virus activation, apoptosis of T cells, and an increased risk of
developing certain types of cancer.
In accordance with the invention, the dose or dosage regimen comprising a
prophylactically or therapeutically effective amount of one or more anti-CD3
antibody or antigen-binding fragments for the treatment of an mune disorder
may be repeated at 1 month, 2 months, 4 months, 6 months, 8 months, 12 months, 15
months, 18 months or 24 months or longer after the initial or previous dose or dosage
regimen comprising a prophylactically or therapeutically effective amount of one or
more anti-CD3 antibody or antigen-binding fragments. The repeat dose or dosage
regimen may be administered as a matter of course, when symptoms associated with
said autoimmune disorder recur after an improvement following the initial or previous
dose or dosage regimen, or when symptoms ated with said autoimmune disorder
do not improve after the initial dose or dosage regimen of anti-CD3 antibody or
antigen-binding fragments according to methods of the invention. For example, with
respect to diabetes, a repeat dose or dosage n comprising a prophylactically or
therapeutically ive amount of one or more anti-CD3 antibody or antigen-binding
nts may be administered to a subject when, for example, the t's e
daily insulin use at 1 month, 2 , 4 months, 6 months, 8 months, 12 months, 15
months, 18 months or 24 months or longer after initial or previous treatment with
anti-CD3 antibody or antigen-binding nts does not decrease by at least 5%, at
least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least
70%, at least 80% or at least 90% compared to pre-treatment levels. Alternatively,
with respect to diabetes, a repeat dose or a dosage regimen comprising a
prophylactically or therapeutically effective amount of one or more anti-CD3
antibody or antigen-binding fragments may be administered to a subject when, for
e, the subject's HA 1 or HA 1 C levels at 1 month, 2 months, 4 months, 6
w ALLALvau Lnyunnvv; u; LLALLALMLLUUMIJIJLVUULVV auLvLLu LALwJ LVL’MLI/ LL; tjvllvuu u;
remission or disappearance of active disease. Immunosuppressive agents used for
months, 8 months, 12 months, 15 months, 18 months or 24 months or longer after
initial or previous treatment with anti-CD3 antibody or antigen-binding fragments do
not decrease by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at
least 50%, at least 60%, at least 70%, at least 80% or at least 90% compared to pre-
ent levels. In another embodiment, with respect to diabetes, a repeat dose or
dosage n comprising a prophylactically or therapeutically effective amount of
one or more anti-CD3 antibody or n-binding fragments may be administered to
a subject when, for example, the subject's C-peptide response at 1 month, 2 months, 4
months, 6 months, 8 months, 12 months, 15 months, 18 months or 24 months or
longer after initial or us treatment with anti-CD3 antibody or antigen-binding
fragments decreases by more than 5%, more than 10%, more than 20%, more than
%, more than 40%, more than 50%, more than 60%, more than 70%, more than
80% or more than 90% compared to eatment levels.
Autoimmune es are non-infectious immunological es caused by
immune ses that are directed to normal components of human cells, tissues and
organs. mune diseases are often chronic diseases that gradually erode ed
tissues and organs. Common es now classified as autoimmune diseases due to
the presence of inappropriate autoimmune responses include type I insulin-dependent
diabetes, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple
sclerosis (MS), inflammatory bowel disease (IBD); myasthenia gravis, celiac’s
e, Sjogren's me, Grave’s disease, Crohn’s disease, autoimmune hepatitis,
psoriasis, psoriatic tis, asthma, allergic rhinitis, effects from organ
transplantation, or graft vs. host disease (GVHD) and us other diseases
involving an inflammatory immune response.
Because autoimmune diseases are typically chronic, they generally require
lifelong treatment and monitoring. Conventional therapies for autoimmune disease
are therefore primarily directed to managing the consequences of inflammation
caused by the disease, and only a few autoimmune diseases can be cured or made to
disappear with such treatment. For some autoimmune diseases, administering one of
a limited number of immunosuppressive medications may result in periods of
remission or disappearance of active disease. Immunosuppressive agents used for
LLLuwvv pus/LL LLALLALMLLUUMIJIJL vuuLuLL VJ L Lb tJWI/LLUbVLLLU A UVLLL’ “LL“ LLLuMVLLLb
regulatory T cells (WO 2007/117600; St. Clair E.W. (2009) “Novel Targeted
adjunct therapy e substances that suppress cytokine production, down-regulate
or suppress self-antigen expression or mask major histocompatibility (MHC) antigens.
suppressive medications include anti-inflammatory drugs (e.g., a nonsteroidal
anti-inflammatory drug (“NSAID”), cyclophosphamide, bromocryptine cyclosporine
A, methotrexate, steroids such as glucocorticosteroids and cytokines or cytokine
receptor antagonists. Patients are rarely able to discontinue these immunosuppressive
medications as their mune disease y reappears when medication is
discontinued. Autoimmune disease may become refractive to treatment when
immunosuppressive medications are continued long term and may require ever
increasing doses of suppressive agents.
] Therapeutic antibodies directed to CD3 are believed to e fewer long-
term side effects than many of the immunosuppressive chemotherapies that are
presently available for autoimmune es (). However, prior
antibody based therapies have been found to be problematic, particularly where
repeated administration was employed. Anti-lymphocyte therapies, such as
antilymphocyte globulin (ALG), and monoclonal antibodies directed to B cells, such
as rituximab (Rituxin®) and alemtuzumab (CAMPATH®) reduce circulating and
tissue B cell populations in treated subjects. However, these therapies also cause
severe suppression, which is undesirable for the long term treatment of a
chronic autoimmune disease. The principal complication of severe immune-
suppressive therapy is infection. Systemic immunosuppression can also be
accompanied by undesirable toxic effects and a ion in levels of ietic
stem cells. In addition, patients receiving antibody therapies often develop significant
levels of human anti-mouse antibodies (HAMA), human anti-chimeric antibodies
(HACA) and anti-idiotypic responses, which may limit repeated treatments when a
remission ends.
As discussed above, antibodies directed to ns of the T cell, such as the
T-cell receptor complex (TCR), have been ted as possible therapeutics for the
immunosuppression of autoimmune disease. Anti-CD3 dies are believed to
induce such immunosuppression by reducing pathogenic T cells and inducing
regulatory T cells (WO 2007/117600; St. Clair E.W. (2009) “Novel Targeted
u UVLL “LLuLbVLL.
ies for Autoimmunity,” Curr. Opin. Immunol. 21(6):648-657; Ludvigsson, J.
(2009) “The Role of Immunomodulation Therapy in Autoimmune Diabetes,” J.
Diabetes Sci. l. 3(2):320-330). Anti-T cell antibodies, including anti-CD3,
have therefore been used to influence logical status in a subject by
suppressing, ing or redirecting T cell responses to an antigen. In particular,
Teplizumab, also known as hOKT3yl(Ala-Ala) (containing an alanine at positions
234 and 235) (MacroGenics, Inc.) is an anti-CD3 antibody that had been engineered
to alter the fianction of the T lymphocytes that mediate the destruction of the insulin-
producing beta cells of the islets of the pancreas. Teplizumab binds to an epitope of
the CD38 chain expressed on mature T cells and by doing so.
Due in part to their cross-reactivity with non-human CD3 (which permits
more accurate and responsive ), the anti-CD3 antibodies of the present
ion are considered to have particular utility in the treatment of autoimmune
disease notwithstanding the apparent failures of the prior art.
Such antibodies and their n binding fragments may be used alone or in
conjunction with other pharmacological agents. In ular, the present invention
contemplates therapies involving the administration of such antibodies or antigen
binding fragments in conjunction with anti-B cell antibodies (or antigen-binding
fragments thereof). Anti-B cell antibodies are known in the art (see, WC
2007/117600; ; ; United States s Nos.
,500,362 and 5,736,137; US. Patent Publications Nos. 2003/0219433;
2005/0271658; 271658; 281817; 2006/024295; 2006/024300 and
2006/034835; Clark, EA. et al. (1985) “Role Of The Bp35 Cell Surface Polypeptide
In Human B-Cell Activation,” Proc. Natl. Acad. Sci. (USA) 82(6):1766-1770; Press,
O.W. et al. (1987) “Monoclonal Antibody IF5 (Anti-CD20) Serotherapy ofHuman B
Cell Lymphomas,” Blood 69:584-591). Such conjunctive administration may be
accomplished using joint administration of distinct antibodies or antigen-binding
fragments thereof, or by forming bispecif1c antibodies, or more preferably, by forming
DARTTM diabodies, as described above, having the ability to bind to both CD3 and a
B cell antigen.
combination with an dy targeted to rferon for treatment of a subject
having multiple sclerosis.
Preferably the employed anti-B cell antibody or antigen-binding fragment
will be directed to a B cell surface marker, such as a marker selected from CD19,
CD20, CD22, CD23, CD32B, CD40, B7-l (CD80), B7-2 (CD86), CD79a, CD79b,
CD38, CD27, a lymphocyte fianction-associated antigen (LFA), such as LFA-I or
LFA-3, CFA-I, or another accessory molecule involved in the T cell, B cell
association that leads to T cell and B cell activation in an adaptive immune response.
In a fiarther preferred embodiment, the anti-B cell antibody may be a B cell ing
dy, such as an antibody directed to a marker selected from CD19, CD20, CD22,
CD23, CD32B, CD40, B7-l (CD80), B7-2 (CD86), a lymphocyte function-associated
antigen (LFA), such as LFA-I or LFA-3, CFA-I, or an accessory molecule involved in
the T cell, B cell association.
Alternatively, such combination therapy may comprise administration of an
anti-CD3 antibody or antigen-binding nt thereof, in combination with an
antibody (or antigen-binding fragment thereof) that recognizes an n present on
an antigen presenting cell (e.g., B7-H3). In a still further preferred embodiment, the
combination therapy comprises administration of an anti-CD3 antibody (or n-
binding fragment thereof) in combination with an antibody (or antigen-binding
fragment f) that recognizes a polypeptide involved in B cell activation (either
directly or indirectly) or an immunomodulator such as a member of TNF cytokine
family, or an interferon (e.g., or, B or y interferon). As is understood by those of skill
in the art, such erons are involved in the regulation of proteins that work
together in antigen processing and presentation. These nes stimulate cells to
increase their expression of HLA class I heavy chains. In one preferred embodiment,
the ation therapy comprises administering to a t having active
autoimmune disease an antibody to a T cell antigen in combination with an antibody
to B—interferon. In a fiarther preferred embodiment, the combination therapy
comprises administering to a subject an antibody targeted to a T cell antigen in
combination with an antibody selected from rferon antibodies AVONEX®,
BETASERON® and REBIF®. In a further embodiment, the combination therapy
comprises stering to a subject an antibody targeted to a T cell antigen in
combination with an antibody targeted to B-interferon for treatment of a subject
having le sclerosis.
A subject rece1v1ng the combination therapy also may have reduced ‘l‘ cell responses
to autoantigens as detected by in vitro by proliferation or cytokine production assays
In a further embodiment, the anti-CD3 antibody may be a non-mitogenic
antibody or a reduced-mitogenic antibody that inhibits or prevents T cell activation
when a T cell comes in contact with its specific n on an antigen presenting cell,
in particular an antigen presenting B cell. As used herein, the term “non-mitogenic T
cell antibody” means an antibody that is engineered by ng the Fc receptor of the
antibody such that it does not trigger the initial activation events and ensuing release
of cytokines that are seen when a T cell is activated. A “reduced mitogenic T cell
dy” is an antibody c for a T cell antigen that reduces the initial activation
events and release of cytokines that occur when a T cell is activated. The non-
mitogenic or d mitogenic antibody may be useful for preventing initial “first
dose side effects” seen when an anti-lymphocyte antibody is administered to patient.
The non-mitogenic or reduced mitogenic antibody may be an ered antibody
having a modified Fc nt that prevents or inhibits binding by effector cells.
C. Methods of Administration
In one , embodiments of the present invention provide treated human
subjects so as to achieve and maintain clinical remissions for longer periods than
remissions achieved by subjects d with a conventional therapy. For example,
where a conventional y achieves a remission of symptoms of an autoimmune
disease for three months, the compositions of the present invention may provide a
complete remission of symptoms of up to six months, up to 12 months and in some
cases up to one to two years or longer. It is plated that for certain autoimmune
diseases it may be possible to provide a complete remission that does not e,
particularly where therapy begins shortly after the autoimmune disease is sed.
The clinical ion achieved with the ation therapy may be a
complete remission, or it may be a partial remission in which significant reductions in
disease ms are maintained for an extended period. For example, a subject
receiving the therapy of the present invention may have d mune
responses as determined by reduced levels of detectable autoantibodies in body fluids
and tissues, for example in cerebrospinal fluid (CSF), serum, urine or in body tissues.
A subject receiving the combination therapy also may have reduced T cell responses
to autoantigens as detected by in vitro by proliferation or cytokine production assays
desired clinical response. s for determining the dosage of a therapeutic
using peripherial blood mononuclear cells (PBMCs) or purified T cells when
compared with subjects d with a conventional therapy.
The itions of the present invention may be administered by any
suitable means, including parenteral, topical, subcutaneous, intraperitoneal,
intrapulmonary, intranasal, and/or intralesional administration. eral infilsions
include intramuscular, intravenous, rterial, intraperitoneal, or subcutaneous
administration. In addition, the antibody may suitably be administered by pulse
infilsion, e.g., with increasing doses of the antibody. Preferably, the dosing is given
intravenously or subcutaneously, and more preferably by intravenous infusion(s).
Each exposure may be provided using the same or a different administration means.
In one embodiment, each exposure is by intravenous administration. In another
embodiment, each exposure is given by subcutaneous administration. In yet another
embodiment, the exposures are given by both intravenous and aneous
administration.
In one embodiment, the therapeutic antibodies are administered as a slow
enous on which may commence at a rate hour to deliver the molecules of
the invention in imately 15 minutes to 4 hours. However, if the subject is
experiencing an infiJsion-related reaction, the infusion rate is preferably reduced, e. g.,
to half the current rate. The treated subjects may receive a prophylactic treatment of
acetaminophen/paracetamol (e.g., about lg) and diphenhydramine HCl (e.g., about 50
mg or equivalent dose of similar agent) by mouth about 30 to 60 s prior to the
start of an infilsion.
The therapy provided by combination compositions of the present invention
(including DARTTM diabodies) may be stered to a subject using an initial dose
of first antibody that is less than the amount of such antibody needed to achieve a
clinical response in therapy for an autoimmune disease when administered as a single
antibody therapy. A dose of a therapeutic anti-T cell antibody that is less than the dose
needed to e depletion of T cells that are able to ize and respond to
autoantigens in a therapy providing a single antibody may be sufficient to provide a
desired clinical se. Methods for determining the dosage of a therapeutic
pharmaceutical art in the form of lyophilized formulations or aqueous solutions.
antibody needed to achieve a clinical response are known to those of skill in the art.
For example, a clinical response in the subject may be ed as time to disease
ssion, reduction of clinical symptoms, reduction in levels of laboratory
s, reduction in the need for retreatment, or by any other clinical means
ized as a useful indicator of improvement in status of the autoimmune disease.
The second antibody of a combination therapy may also be administered to a
subject in need of treatment as an initial dose that is less than an effective dose for
achieving a clinical response when the antibody is administered alone. For example,
doses of a depleting anti-B cell antibody that achieve less than 100% B-cell depletion,
less than 50% B cell depletion, less that 30% depletion or even no B cell depletion
may be administered together with a first anti-T cell antibody to achieve a clinical
se that provides suppression of an immune response to an autoantigen equal to,
or better than the clinical response achieved by administering an amount of a B cell
depleting antibody that provides 100% depletion of B cells in the subject when
administered alone.
In some instances, clinical response may be a response that neither the first
nor the second antibody achieves when administered alone. In other instances, the
clinical response may be equivalent to that ed by stration of a single
antibody therapy, where the combination therapy provides less suppression of
a treated t’s immune system than a single antibody therapy. In one preferred
embodiment, the synergistic response provided by the ation therapy reduces or
eliminates a subject’s se to an autoantigen while providing lower levels of
immunosuppression. General immunosuppression is a significant m for
previously available antibody therapies.
D. Pharmaceutical Formulations
Therapeutic formulations of the antibodies used in embodiments of the
present invention are prepared for storage, shipment and administration by mixing a
composition of the present invention having a d purity with optional
pharmaceutically able carriers, excipients or stabilizers recognized in the
pharmaceutical art in the form of lyophilized formulations or aqueous solutions.
LJutJLLLLLuvu yuvvuv; u; vkuv; Lva vuLLVVLLuquv LL; w LLVLLLLVULUWLLJ vaLvu UULLUMLLLVL ”mun;
QC on amnnnlp nr aanhpfl'p inr‘linafinn- Hap nuanfifv n‘F anfivn arr-sni- ‘Xnmarp Hop
The term “pharmaceutically acceptable” means approved by a regulatory
agency of the Federal or a state government or listed in the US. Pharmacopeia or
other generally recognized pharmacopeia for use in animals, and more particularly in
humans. The term “carrier” refers to a diluent, nt (e.g., Freund’s adjuvant
ete and incomplete), excipient, or vehicle with which the therapeutic is
administered. Such ceutical carriers can be sterile liquids, such as water and
oils, including those of petroleum, , vegetable or synthetic origin, such as
peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred
carrier when the pharmaceutical composition is administered intravenously. Saline
ons and aqueous dextrose and ol solutions can also be employed as liquid
carriers, ularly for injectable solutions. Suitable pharmaceutical excipients
include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium te, ol monostearate, talc, sodium de, dried skim milk,
glycerol, propylene, glycol, water, ethanol and the like. The ition, if desired,
can also contain minor amounts of wetting or emulsifying agents, or pH buffering
agents. These compositions can take the form of solutions, suspensions, on,
tablets, pills, capsules, s, sustained-release formulations and the like.
] The compositions of the invention include bulk drug compositions useful in
the manufacture of pharmaceutical compositions (e.g, impure or non-sterile
compositions) and pharmaceutical compositions (z'.e., compositions that are suitable
for administration to a subject or patient) which can be used in the preparation of unit
dosage forms. Such compositions comprise a prophylactically or therapeutically
effective amount of a prophylactic and/or therapeutic agent sed herein or a
combination of those agents and a pharmaceutically acceptable carrier. Preferably,
compositions of the invention comprise a prophylactically or therapeutically effective
amount of humanized antibodies of the invention and a pharmaceutically acceptable
carrier.
Generally, the ingredients of compositions of the invention are supplied
either separately or mixed together in unit dosage form, for example, as a dry
lyophilized powder or water free concentrate in a hermetically sealed container such
as an ampoule or sachette indicating the quantity of active agent. Where the
composition is to be administered by infusion, it can be dispensed with an infiJsion
bottle containing sterile pharmaceutical grade water or saline. Where the composition
is administered by ion, an ampoule of sterile water for injection or saline can be
provided so that the ingredients may be mixed prior to administration.
Pharmaceutical compositions suitable for injection include sterile aqueous solutions
where the active agents are water soluble, or dispersions or sterile powders for
oraneous preparation of e injectable solutions. Compositions for use in
the combination therapy may be prepared by incorporating the active antagonist or
antibody in the required amount with appropriate carriers, for example water, ethanol,
polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol) and suitable
mixtures thereof. Isotonic agents such as sugars, polyalcohols such as mannitol,
sorbitol or sodium chloride may be included in the composition.
The compositions of the invention can be formulated as neutral or salt forms.
Pharmaceutically acceptable salts e, but are not limited to, those formed with
anions such as those derived from hydrochloric, oric, acetic, oxalic, tartaric
acids, etc., and those formed with cations such as those derived from sodium,
potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-
ethylamino ethanol, histidine, ne, etc.
E. Kits
The ion provides a pharmaceutical pack or kit comprising one or more
containers filled with humanized antibodies of the invention. onally, one or
more other prophylactic or therapeutic agents useful for the treatment of a disease can
also be included in the ceutical pack or kit. The invention also provides a
pharmaceutical pack or kit comprising one or more containers filled with one or more
of the ingredients of the pharmaceutical compositions of the invention. Optionally
associated with such container(s) can be a notice in the form prescribed by a
governmental agency regulating the cture, use or sale of pharmaceuticals or
biological products, which notice reflects al by the agency of manufacture, use
or sale for human administration.
The present invention provides kits that can be used in the above methods.
In one embodiment, a kit comprises one or more humanized antibodies of the
invention. In another embodiment, a kit fiarther comprises one or more other
prophylactic or eutic agents useful for the treatment of cancer, in one or more
containers. In another embodiment, a kit fiarther comprises one or more cytotoxic
antibodies that bind one or more cancer antigens associated with cancer. In certain
ments, the other prophylactic or therapeutic agent is a chemotherapeutic. In
other embodiments, the prophylactic or therapeutic agent is a biological or hormonal
therapeutic.
] In one embodiment, the present invention es an article of manufacture
containing antibodies to be used for the combined therapy for treatment of
autoimmune disease. The article of manufacture ses a container sing a
first antibody that binds an antigen present on a T cell and a pharmaceutically
able carrier, excipient or diluent within the container. The article of
manufacture further comprises a second container sing a second antibody
directed to a B cell surface marker and a pharmaceutically acceptable carrier,
excipient or diluent and instructions for administering the composition to a subject in
need of treatment for mune disease. Where the first and second antibodies are
determined to be complementary and to not adversely affect each other, the first and
the second antibody may be provided in a single container containing the first and
second antibody in appropriate concentrations for administration together with a
package insert and instructions for administration.
Containers of the article of cture may be of any suitable material that
will not react with or otherwise affect the preparation. The article of manufacture may
r comprise a second or a third container comprising a pharmaceutically-
acceptable diluent buffer, such as iostatic water for injection, phosphate-
buffered saline, Ringer's on and dextrose solution. The article of manufacture
may also include other material that may be desired from a cial and user
standpoint including other buffers, diluents, filters, needles and syringes.
L/Alj1L/DD1U11. no uovu 11V1L/111, 111L/L11UUD 1U1 cuuuls u1u511UD1D 11D L11CLL L11L/DL/ 111L/L11UUD
assist in making a clinical determination regarding the classification, or nature, of
VII. Diagnostic Methods Using the Anti-CD3 Antibodies of the
Present invention
Antibodies to CD3 made by the methods sed herein may also be used
to identify the presence or absence of cancerous cells, or the level thereof, which are
circulating in blood after their release from the cell surface (e.g., soluble CD3). Such
circulating antigen may be an intact CD3 antigen, or a fragment thereof that retains
the ability to be detected according to the methods taught herein. Such detection may,
for example, be effected by FACS is using standard methods commonly used in
the art.
In a preferred embodiment of the diagnostic methods of this invention, the
antibody bears a detectable label. Examples of labels that may be used include a
radioactive agent (e.g., Scandium-47, Technetium-99m, -l l l, Iodine-l3l,
Rhenium-186, Rhenium-188, Samarium-153, Holmium-166, Lutetium-l77, Copper-
64, Scandium-47, Yttrium-900), an enzyme or a fluorophore, such as phycoerythrin or
fluorescein isothiocyanate (also known as fluoroisothiocyanate or FITC).
One method of using the antibodies for diagnosis is in vivo tumor imaging
by linking the antibody to a radioactive or radio-opaque agent, administering the
antibody to the individual and using an x-ray or other imaging machine to visualize
the localization of the labeled antibody at the surface of cancer cells expressing the
antigen. The antibody is administered at a concentration that promotes binding at
physiological conditions.
In vitro techniques for detection of CD3 are routine in the art and include
enzyme linked sorbent assays (ELISAs), precipitations,
immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and
Western blot is.
] The invention also provides s of aiding diagnosis of cancer
characterized by cancer cells that express CD3 in an individual using any dy
that binds to CD3 and any other methods that can be used determine the level of CD3
expression. As used herein, s for g sis” means that these methods
assist in making a clinical determination regarding the classification, or nature, of
LLLLLLWUVLJ vu n v; “Lukuv LLLULMuLLLb mnvvxwun v v vuLLuLu MAL“ VLULLLL u “Lukuv,
myasthenia gravis, lupus, toid arthritis, , acute iuvenile onset diabetes,
cancer, and may or may not be conclusive with respect to the definitive diagnosis.
Accordingly, a method of aiding sis of cancer can comprise the step of
detecting the level of CD3 in a biological sample from the individual and /or
determining the level of CD3 expression in the sample. dies recognizing the
antigen or a portion thereof may also be used to create stic assays for
ing antigen released or secreted from living or dying cancer cells in bodily
fluids, including but not limited to, blood, saliva, urine, pulmonary fluid, or ascites
fluid. The anti-CD3 antibodies made by the methods disclosed herein may be used to
determine whether an individual diagnosed with cancer may be deemed a candidate
for immunotherapy using antibodies directed against CD3. In one embodiment, a
biopsy sample may be tested for expression of CD3, using antibodies directed against
CD3. Individuals with cancer cells that express CD3 are suitable candidates for
immunotherapy using antibodies directed against CD3. Staining with anti-CD3
antibody may also be used to distinguish cancerous tissues from normal tissues.
s of using anti-CD3 antibodies for diagnostic purposes are useful
both before and after any form of anti-cancer treatment, e.g., chemotherapy or
radiation therapy, to determine which tumors are most likely to respond to a given
treatment, prognosis for dual with cancer, tumor subtype or origin of metastatic
disease, and progression of the disease or response to treatment.
The compositions of this invention are particularly suitable for the diagnosis
of disease states other than cancer, using the methods generally described above in
ation with other diseased (non-cancerous) cells. Disease states suitable for use
in the methods of this invention include, but are not d to, diseases or disorders
ated with inflammatory or autoimmune responses in individuals. The methods
described above may be used for modulating inflammatory or autoimmune ses
in individuals. Diseases and conditions resulting from inflammation and autoimmune
disorders that may be subject to diagnosis and /or treatment using the compositions
and methods of the invention include, by way of illustration and not of limitation,
multiple sclerosis, meningitis, encephalitis, stroke, other cerebral traumas,
inflammatory bowel e including ulcerative colitis and Crohn's disease,
myasthenia , lupus, rheumatoid arthritis, asthma, acute juvenile onset diabetes,
“Lulu ALLVL- J\U/-AVV./ Avxu, L VA A “VLvauLuLL vv v vau/ v1 Ajvul. VLLuvLLLLLu
hvnnthesis, the CSCs provide a small, distinct subset of cells within each tumor that
AIDS dementia, atherosclerosis, nephritis, tis, atopic dermatitis, psoriasis,
myocardial ischemia and acute leukocyte-mediated lung injury.
Still other indications for diagnostic and/or therapeutic use of dies and
other therapeutic agents of the ion include administration to individuals at risk
of organ or graft rejection. Over recent years there has been a considerable
improvement in the efficiency of surgical techniques for transplanting tissues and
organs such as skin, kidney, liver, heart, lung, pancreas and bone marrow. Perhaps the
principal outstanding problem is the lack of satisfactory agents for ng
immunotolerance in the ent to the transplanted aft or organ. When
allogeneic cells or organs are transplanted into a host (i.e., the donor and donee are
different individuals from the same species), the host immune system is likely to
mount an immune response to foreign antigens in the lant (host-versus-graft
disease) g to destruction of the transplanted .
Monoclonal antibodies to CD3 made by the methods disclosed herein may
be used to identify the presence or absence of human cancer stem cells in a variety of
tissues. Cancer stem cells (CSCs) have been hypothesized to play a role in tumor
growth and metastasis (Ghotra, V.P. et al. (2009) “The Cancer Stem Cell
Microenvironment And Anti-Cancer Therapy,” Int. J. . Biol. 85(11):955-962;
Gupta, P.B. et al. (2009) “Cancer Stem Cells: Mirage 0r Reality?” Nat. Med.
lS(9):lOlO-lOl2; Lawson, J.C. et al. (2009) “Cancer Stem Cells In Breast Cancer
And Metastasis,” Breast Cancer Res. Treat. 118(2):241-254; Hermann, P.C. et al.
(2009) “Pancreatic Cancer Stem Cells--Insights And Perspectives,” Expert Opin.
Biol. Ther. 9(10):127l-l278; Schatton, T. et al. (2009) “Identification And Targeting
OfCancer Stem Cells,” Bioessays 31(10):lO38-1049; Mittal, S. et al. (2009) r
Stem Cells: The Other Face OfJanus,” Amer. J. Med. Sci. 338(2):lO7-ll2; Alison,
M.R. et al. (2009) “Stem Cells And Lung Cancer: Future Therapeutic s?”
Expert Opin. Biol. Ther. 9(9):1127-1141; Charafe-Jauffret, E. et al. (2009) “Breast
Cancer Stem Cells: Tools And Models To Rely 0n,” BMC Cancer 9:202; Scopelliti,
A. et al. (2009) “Therapeutic Implications Of Cancer Initiating Cells,” Expert Opin.
Biol. Ther. 9(8):1005-1016; PCT Publication WO 2008/091908). Under this
hypothesis, the CSCs provide a small, distinct subset of cells within each tumor that
human mammalian species. Additionally, the binding of the chimeric mAbl antibody
xxron nnmnornr] fn flnof AP 014 onfdlanrltr nnmnnnor‘ AP {-140 Inn/“0141.701: Y7OT‘1‘O14" 1m Ala1 T F f)
are capable of indefinite self-renewal and of developing into the more adult tumor
cell(s) that are relatively limited in replication capacity. It has been esized that
these cancer stem cells might be more resistant to chemotherapeutic agents, radiation
or other toxic conditions, and thus, persist after clinical ies and later grow into
secondary , metastases or be responsible for relapse. It has been suggested that
CSCs can arise either from 'normal' tissue stem cells or from more differentiated
tissue progenitor cells.
Uses described in this application that recite their use for anti-CD3
antibodies also encompass the use of other CD3 agonists, antagonists and tors
as bed herein for the use of identification and treatment of cancer stem cells. In
such embodiments, anti-CD3 antibodies and other CD3 agonists, antagonists and
modulators are used for identification, diagnosis or therapeutic treatment of cancer
stem cells using similar methods described, and alterations within the scope of the
ordinary skilled practitioner are made to tailor the method to the identification
/diagnosis or treatment of cancer stem cells.
Having now generally described the invention, the same will be more y
understood through reference to the following examples, which are provided by way
of illustration and are not intended to be limiting of the present invention unless
specified.
mAbl Binds to Both Human and Cynomolgus Monkey CD3
In order to assess the ability of mAbl to bind to human CD3, a capture
ELISA was med. Plates were coated with l ug/ml of soluble lgus CD3
(“sCD3”) and incubated in the ce of various concentrations of a chimeric
variant of mAbl dy (ch-mAbl) (containing the variable sequences of mAbl
and the constant regions of a human antibody), a humanized variant (h-mAbl) and an
antibody composed of the light chain of the chimeric mAbl antibody and the heavy
chain of the humanized variant of mAbl. The s of this experiment are shown in
Figure 1A. The experiment shows the ability of mAbl to bind to the CD3 of a non-
human mammalian species. Additionally, the binding of the chimeric mAbl antibody
was ed to that of an antibody composed of the humanized variant mAbl LC-2
aaccgagttt iiiiiiiiiiiiiiiiiiiiiactctgacca tttccagcct gcagcctgaa gatttcgcaa
and the heavy chain of mAbl. The s of this experiment are shown in Figure 1B.
The experiment shows the ability of mAbl to bind to human CD3. Figures 1A and
1B thus reveal that the humanized mAbl was e of binding to both human CD3
and a CD3 of a non-human mammal. Humanized mAb showed binding to sCD3 and
hCD3 that was r to that of the ic mAbl.
Example 2
Humanization of mAbl
Humanized tives of mAbl were prepared. The amino acid sequences
and encoding polynucleotide sequences of these humanized derivatives are shown
below. The CDRs are shown in boldface and underlined.
Amino Acid Sequence of Humanized mAbl Variable Light Chain Variant l
(SEQ ID NO:10):
DIQ TQSPSS LSASVGDRVT :TCSASSSVS YMNWYQQKPG KAPKRLIY2§
SKLASGVPSR GT*E TLT SSLQPfl DEATYYCQQW SRNPPTFGGG
TKVfl K
Polynucleotide Sequence Encoding Humanized mAbl Variable Light Chain
Variant 1 (SEQ ID NO:11):
caga agtc cccc:ccagc ch c Lngggcga
cagagtgaca aLcachg L ccgccachc chchchc L
ggLaLcagca gaagcccggc aaggccccca agcgchgaL cLacgachc
tccaagc:gg cctccggch gccceccaga ch c
caccgagLLc accctgacca :ctccagcct gcagcccgag gacttcgcca
chacLach ccagcagtgg :cccggaacc cccctacct: cggcggaggc
accaaggtgg aaatcaag
Amino Acid Sequence of Humanized mAbl Variable Light Chain Variant 2
(mAbl LC-2) (SEQ ID NO:12):
DVV TQSPA" MSAhPGflKVT TCSASSSVS YMNWYQQKPG KAPKRWIY2§
SKLASGVPSR FSGSGSGTflh TLT SSLQPfl DEATYYCQQW SRNPPTFGGG
TKVfl K
Polynucleotide ce Encoding Humanized mAbl Variable Light Chain
Variant 2 (SEQ ID NO:13):
gachgnga Lgacccach aLc aLgangcLL Lcccaggcga
gaaagtgacc aLLacaLch chcLLccag chLngLcc LacaegaacL
ggtatcagca gaagccaggg aaagcaccca agaggeggae cLacgachc
cegg chccggcg: gccaagccgg 'LchngLa ngchcaggL
aaccgagLLL achLgacca t:tccagcct gcagcctgaa gatttcgcaa
(h-mAb2 VL-2) (SEQ ID NO:18):
ca sacLa ng Lcagcagtgg tccagaaatc cccctacatt tggcggaggg
ac saaag egg aaatcaag
] Amino Acid Sequence of Humanized mAbl Variable Heavy Chain (SEQ ID
NO:14):
QVQLVQSGAE VKKPGASVKV SCKASGYTFT RSTMHWVRQA PGQGLnW GI.|.
INPSSAYTNY NQKFKDRVT: TADKSTSTAY MELSSLRSED TAVYYCASBQ
VHYDYNGFPY WGQGTLVTVS S
Polynucleotide ce Encoding Humanized mAbl Variable Heavy Chain
Chain (SEQ ID NO:15):
caggtgcagc sggsgcagsc ngcgccgaa gtgaagaaac ctggcgcctc
cgcgaaggvg vcccgcaagg cc:ccggcta caccttcacc cggtccacca
tgcactggg: gcgacaggcc ccaggccagg aatg gatcggc:ac
atcaacccc: ccagcgcc:a caccaac:ac aaccagaaat tcaaggaccg
cgvgaccasc accgccgaca agtccaccag caccgchac acggaacvgv
csagcccgcg gagcgaggac accgccgtgt actactgcgc thcccccag
gcgcacvacg acgg cLsccccsac ngggccagg tgg:
gacagtgtcc tcc
Example 3
Humanization of mAb2
Humanized derivatives of mAb2 were prepared. The amino acid sequences
and encoding polynucleotide sequences of these humanized derivatives are shown
below. The CDRs are shown in boldface and underlined.
Amino Acid Sequence of Humanized mAb2 Variable Light Chain Variant l
(h-mAb2 VL-l) (SEQ ID :
QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWFQQ KPGQAPRTL:
APWT PARFSGSLLG GKAALTITGA DYYC ALWYSNLWVF
GGGTKLTVLG
Polynucleotide ce Encoding Humanized mAb2 Variable Light Chain
Variant l (h-mAb2 VL-l) (SEQ ID NO:17):
Lgachagga gccttcactg tccc caggcggaac
agaL ccagcacagg cgcagtgacc acatc:aact
gchcagcag aagccaggac aggcaccaag gasc
acaaaagggc cccc cctgcacggs Lchngaag
sgcsgggc ggaaaggccg ggca caggccgagg
acgaagccga LeacLangL aLagcaaLcs gagggsgLsc
gggggtggca caaaac,gac schggga
Amino Acid Sequence of Humanized mAb2 Variable Light Chain Variant 2
2 VL-2) (SEQ ID NO:18):
ggggguggca UddddULgdU LngUnggd
QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRTL:
GGTNKRAPWT PARFSGSLLG GKAALTITGA QAfiDflADYYC ALWYSNLWVF
GGGTKLTVLG
Polynucleotide Sequence Encoding Humanized mAb2 Variable Light Chain
Variant 2 (h-mAb2 VL-2) (SEQ ID NO:19):
caggcdgdgg Lgachagga gccttcactg accgtgtccc caggcggaac
,g,gaccc:g acatgcagat cagg cgcagtgacc acatc:aact
gngcagcag aagccaggac aggcaccaag gaccc,ga,c
acaaaagggc ,ccc,ggacc cctgcacggc L,Lchgaag
,c,gc,gggc ggaaaggccg ctctgacta: ggca caggccgagg
acgaagccga LLacLachL gc,c,nggc aLagcaaLcc gdgggdngc
gggggtggca caaaac,gac dgdchggga
Amino Acid Sequence of zed mAb2 Variable Light Chain Variant 3
(h-mAbZ VL-3) (SEQ ID NO:20):
QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT WFQE KPGQAPRTL:
GGTNKRAPWT PARFSGSLLG GKAALTITGA QAfiDflADYYC ALWYSNLWVF
GGGTKLTVLG
Polynucleotide Sequence Encoding Humanized mAb2 le Light Chain
Variant 3 (h-mAb2 VL-3) (SEQ ID NO:21):
caggcdgdgg gga actg accgtgtccc caggcggaac
,g,gaccc:g acatgcagat ccagcacagg cgcagtgacc acatc:aact
chccaggag aagccaggac aggcaccaag ga,c
acaaaagggc ,ccc,ggacc cctgcacggc L,Lchgaag
,c,gc,gggc ggaaaggccg ctctgacta: ggca caggccgagg
acgaagccga chL gcvcvgtggv aLagcaaLcc gvgggngvc
ggca caaaac,gac dgdchggga
Amino Acid Sequence of Humanized mAb2 le Light Chain Variant 4
(h-mAbZ VL-4) (SEQ ID NO:22):
QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWFQQ KPGQAPRGL:
GGTNKRAPWT PARFSGSLLG GKAALTITGA QAfiDflADYYC ALWYSNLWVF
GGGTKLTVLG
Polynucleotide Sequence Encoding Humanized mAb2 Variable Light Chain
Variant 4 (h-mAb2 VL-4) (SEQ ID NO:23):
Lgachagga gccttcactg tccc caggcggaac
acatgcagat ccagcacagg cgcagtgacc acatc:aact
chccagcag aagccaggac aggcaccaag gggccvgavc
acaaaagggc ,ccc,ggacc cctgcacggc L,Lchgaag
,c,gc,gggc ggaaaggccg ctctgacta: taccggggca caggccgagg
acgaagccga L,acLachL gcvcvgtggv aLagcaaLcc gdgggdngc
gggggtggca ,gac dgdchggga
gggggtacaa acaaaagggc tccctggacc cctgcacggt gaag
gggc ggaaaggccg ctctgactat taccggggca caggccgagg
Amino Acid Sequence of Humanized mAb2 Variable Light Chain Variant 5
(h-mAb2 VL-S) (SEQ ID NO:24):
QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQE KPGQAPRTL:
GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF
GGGTKLTVLG
Polynucleotide Sequence Encoding Humanized mAb2 Variable Light Chain
Variant 5 (h-mAb2 VL-5) (SEQ ID NO:25):
caggcvgvgg Lgachagga gccttcactg accgtgtccc caggcggaac
,g,gaccc:g acatgcagat ccagcacagg cgcagtgacc acatc:aact
gngcaggag aagccaggac aggcaccaag gaccc,ga,c
acaaaagggc ,ccc,ggacc cctgcacggv L,Lchgaag
vcvgcvgggc ggaaaggccg ctctgacta: taccggggca caggccgagg
acgaagccga LLacLaSLgL gcvcvgtggv aLcS gvgggSgch
ggca caaaaCSgac SgSgCngga
Amino Acid Sequence of Humanized mAb2 Variable Light Chain Variant 6
(h-mAb2 VL-6) (SEQ ID NO:26):
QAVVTQEPSL TVSPGGTVTL GAVT TSNYANWVQQ KPGQAPRGL:
GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC LWVF
GGGTKLTVLG
Polynucleotide Sequence Encoding Humanized mAb2 Variable Light Chain
Variant 6 (h-mAb2 VL-6) (SEQ ID NO:27):
caggcvgvgg Lgachagga gccttcactg accgtgtccc caggcggaac
,g,gaccc:g acatgcagat cagg gacc aact
gngcagcag aagccaggac aggcaccaag gggccvgavc
acaaaagggc ,ccc,ggacc cctgcacggv L,Lchgaag
vcvgcvgggc ggaaaggccg ctctgacta: taccggggca caggccgagg
acgaagccga LLacLaSLgL gcvcvgtggv aLagcaaLcS gch
gggggtggca Sgac SgSgCngga
Amino Acid Sequence of Humanized mAb2 le Light Chain Variant 7
(h-mAb2 VL-7) (SEQ ID NO:28):
QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWFQE KPGQAPRGL:
APWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF
GGGTKLTVLG
Polynucleotide ce Encoding Humanized mAb2 Variable Light Chain
Variant 7 (h-mAb2 VL-7) (SEQ ID NO:29):
caggcvgvgg Lgachagga gccttcactg tccc caggcggaac
,g,gaccc:g acatgcagat cagg cgcagtgacc acatc:aact
acgccaa,,g gLLccaggag aagccaggac aggcaccaag gggcc:gatc
gggggtacaa acaaaagggc tccc:ggacc cctgcacggt tttctggaag
vcvgcvgggc ggaaaggccg ctat taccggggca caggccgagg
caggctgtgg tgactcagga actg accgtgtccc caggcggaac
cctg acatgcagat ccagcactgg agcagtgact acctctaact
acgaagccga LLacLaeLgL gc,chngL aLagcaatct gtgggtgttc
gggggtggca caaaac,gac Lgechggga
Amino Acid Sequence of Humanized mAb2 Variable Light Chain Variant 8
(h-mAb2 VL-8) (SEQ ID NO:30):
QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQE KPGQAPRGL:
GGTNKRAPWT PARFSGSLLG GKAALTITGA QAflDflADYYC ALWYSNLWVF
GGGTKLTVLG
Polynucleotide Sequence Encoding Humanized mAb2 le Light Chain
Variant 8 2 VL-8) (SEQ ID NO:31):
caggcegegg Lgachagga gccttcactg accgtgtccc caggcggaac
,g,gaccc:g acatgcagat ccagcacagg cgcagtgacc acatc:aact
gngcaggag aagccaggac aggcaccaag gggccvgavc
acaaaagggc gacc cctgcacgge L,Lchgaag
vcvgcvgggc ggaaaggccg ctctgacta: taccggggca caggccgagg
acgaagccga eLgL gcvcvgtggv aLagcaaLce gagggegLec
gggggtggca caaaac,gac vgvchggga
Amino Acid Sequence of Humanized mAb2 Variable Light Chain Variant 9
(h-mAb2 VL-9) (SEQ ID NO:32):
QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQE KPGQAFRGL:
GGTNKRAPWT PARFSGSLLG GKAALTITGA QAfiDflADYYC ALWYSNLWVF
GGGTKLTVLG
Polynucleotide Sequence Encoding Humanized mAb2 Variable Light Chain
Variant 9 (h-mAb2 VL-9) (SEQ ID NO:33):
caggcegegg Lgachagga gccttcactg accgtgtccc caggcggaac
,g,gaccc:g acatgcagat ccagcacagg cgcagtgacc acatc:aact
acgccaa,,g gngcaggag aagccaggac aggca,,cag gggccvgavc
gggggtacaa acaaaagggc gacc cctgcacgge L,Lchgaag
vcvgcvgggc ggaaaggccg ctctgacta: taccggggca gagg
acgaagccga LLacLaeLgL gcvcvgtggv aLagcaaLce gagggegLec
ggca ,gac vgvchggga
] Amino Acid Sequence of Humanized mAb2 Variable Light Chain Variant lO
(h-mAb2 VL-lO) (SEQ ID NO:34):
EPSL TVSPGGTVTL TCRSSTGAVT WFQQ KPDHLFTGL:
GGTNKRAPWT PARFSGSLLG GKAALTITGA QAfiDflADYYC ALWYSNLWVF
GGGTKLTVLG
] Polynucleotide Sequence Encoding Humanized mAb2 le Light Chain
Variant lO (h-mAb2 VL-lO) (SEQ ID :
caggctgtgg tgactcagga gccttcactg accgtgtccc caggcggaac
tgtgaccctg acatgcagat ctgg agcagtgact acctctaact
nUlVlULVDIVD WEI-ll. VVUQULLI V J. V00
acgctaattg gttccagcag aagcccgacc achgLLcac egggc,gaec
ggcggaacca acaaaagggc ,ccc,ggacc cctgcacgg, aag
tctgc:gggc ggaaaggccg ctctgacta: taccggggca caggccgagg
acgaagccga L,acLaeLgL gc,c,ngg aLagcaaLce geggg,gL,c
gggggtggca caaaac,gac ,g,chggga
Amino Acid Sequence of Humanized mAb2 Heavy Chain t 1 (h-
mAb2 VH-l) (SEQ ID NO:36):
flVQLVflSGGG LVQPGGSLR; SCAASGFTFS TYAMNWVRQA PGKGLEWVGE
IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSHKT HDTAVYYCAR
HGNFGNSYVS WFAYWGQGT; VTVSS
Polynucleotide Sequence Encoding Humanized mAb2 Variable Heavy Chain
Variant 1 (h-mAb2 VH-l) (SEQ ID NO:37):
gaggtgcagc ,gg,ggaaag cggcggagga ctggtgcagc caggtggcag
ac,g ,c g cacc, aca,acgcca
tgaactgggt Ok—Qk—QFI'OO ,anggc,L ggc: ctggaaagg ggc,ggag,g gg,gggcagg
atcaggtcca ag:acaacaa :atgcaacc ,ac,a,gccg tgaa
gga,agaL,c acaatttccc cgacga,,c ,aaaaacag, c,g,aLc,gc
aga,gaac,c gac: aagacaccg ,a L,gegcaaga
cacggaaact tcggcaach ,achgecc ,gca, a,,gggg,ca
gggcacac:g gtgaccgtg: ccagc
Amino Acid Sequence of Humanized mAb2 Heavy Chain t 2 (h-
mAb2 VH-2) (SEQ ID NO:38):
flVQLVflSGGG LVQPGGSLR; SCAASGFTFN TYAMNWVRQA PGKGLEWVGR
IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSHKT HDTAVYYCAR
HGNFGNSYVS WFAYWGQGT; VTVSS
] Polynucleotide Sequence Encoding Humanized mAb2 Variable Heavy Chain
Variant 2 (h-rnAb2 VH-2) (SEQ ID NO:39):
gaggtgcagc aaag cggcggagga ctggtgcagc caggtggcag
cctgcgac,g ,c ,gcgccg Ok—Qk—QFI'OO ,anggc,L cacc, acatacgcca
tgaactgggt ggc: agg ggc,ggag,g gg,gggcagg
atcaggtcca ag:acaacaa :atgcaacc ,ac,a,gccg ac:cagtgaa
gga,agaL,c acaatttccc cgacga,,c ,aaaaacag, c,gc
aga,gaac,c cc,gaagac: aagacaccg ,a L,gegcaaga
cacggaaact tcggcaach ,achgecc ,gca, a,,gggg,ca
gggcacac:g gtg: ccagc
Amino Acid Sequence of Humanized mAb2 Heavy Chain Variant 3 (h-
mAb2 VH-3) (SEQ ID NO:40):
flVQLVflSGGG LVQPGGSLR; SCAASGFTFS TYAMNWVRQA PGKGLEWVAR
NYAT YYADSVKDRF TISRDDSKNS SHKT HDTAVYYCAR
HGNFGNSYVS WFAYWGQGT; VTVSS
Polynucleotide Sequence Encoding Humanized mAb2 Variable Heavy Chain
Variant 3 (h-mAb2 VH-3) (SEQ ID NO:41):
gaggtgcagc ,gg,ggaaag cggcggagga ctggtgcagc caggtggcag
cctgcgac,g ,c Vgcgccg ,L cacc,,Lch aca,acgcca
tgaactgggt gaggcaggc: ctggaaagg ggc,ggag,g gg,ggccagg
atcaggtcca acaa :atgcaacc vac,avgccg ac:cagtgaa
gga,agaL,c acaatttccc cgacga,vc ,aaaaacag, cag,aLc,gc
aga,gaac,c cc,gaagac: aagacaccg ccg,g,ac,a L,g,gcaaga cacggaaact tcggcaach Ok—Qk—QN'OO ,achgvcc ,gng,gca, av,gggg,ca
gggcacac:g gtgaccgtg: ccagc
Amino Acid Sequence of Humanized mAb2 Heavy Chain Variant 4 (h-
mAbZ VH-4) (SEQ ID NO:42):
flVQLVflSGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVGR
IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSHKT ?DTAVYYCVR
HGNFGNSYVS WFAYWGQGT; VTVSS
Polynucleotide Sequence Encoding Humanized mAb2 Variable Heavy Chain
Variant 4 (h-mAb2 VH-4) (SEQ ID NO:43):
gaggtgcagc ,gg,ggaaag agga ctggtgcagc caggtggcag
ac,g ,c Vgcgccg ,L cacc,,Lch aca,acgcca
tgaactgggt gaggcaggc: agg ggc,ggag,g gg,gggcagg
atcaggtcca ag:acaacaa :atgcaacc vac,avgccg ac:cagtgaa
gga,agaL,c acaatttccc cgacga,vc ,aaaaacag, cag,a,c,gc
aga,gaac,c cc,gaagac: aagacaccg ccg,g,ac,a L,g,g,gaga cacggaaact tcggcaach Ok—Qk—QN'OO ,achgvcc ,gng,gca, av,gggg,ca
ac:g gtgaccgtg: ccagc
Amino Acid Sequence of Humanized mAb2 Heavy Chain t 5 (h-
mAbZ VH-5) (SEQ ID NO:44):
flVQLVflSGGG LVQPGGSLR; SCAASGFTFN VRQA WVAR
IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSHKT YCAR
HGNFGNSYVS WFAYWGQGT; VTVSS
Polynucleotide Sequence Encoding Humanized mAb2 Variable Heavy Chain
Variant 5 (h-mAb2 VH-S) (SEQ ID NO:45):
gaggtgcagc ,gg,ggaaag cggcggagga ctggtgcagc caggtggcag
cctgcgac,g dc,,gcgccg ,anggc,L cacc,,Laac gcca
tgaactgggt gaggcaggc: ctggaaagg ag,g gg,ggccagg
tcca acaa :atgcaacc gccg ac:cagtgaa
gga,agaL,c acaatttccc Ok—Qk—QN'OO cgacga,vc ,aaaaacag, c,gc
aga,gaac,c cc,gaagac: aagacaccg ccg,g,ac,a L,g,gcaaga
cacggaaact tcggcaach ,achgvcc ,gng,gca, av,gggg,ca
gggcacac:g gtgaccgtg: ccagc
cctgagactc tcctgtgcag cctctggatt caac acatacgcta
tgaattgggt ccgccaggct ccagggaagg ggctggagtg ggttgcaagg
Amino Acid Sequence of Humanized mAb2 Heavy Chain Variant 6 (h-
mAb2 VH-6) (SEQ ID NO:46):
SGGG LVQPGGSLRL SCAASGFTFN TYAMNWVRQA PGKGLEWVGR
IRSKYNNYAT YYADSVKDRF SKNS LYLQMNSHKT EDTAVYYCVR
SYVS WFAYWGQGT; VTVSS
Polynucleotide Sequence Encoding Humanized mAb2 Variable Heavy Chain
Variant 6 (h-mAb2 VH-6) (SEQ ID :
gaggtgcagc ,gg,ggaaag cggcggagga ctggtgcagc caggtggcag
cctgcgac,g ,c g ,anggc,L cacc,,Laac acatacgcca
tgaactgggt gaggcaggc: ctggaaagg ggc,ggag,g gg,gggcagg
tcca ag:acaacaa :atgcaacc vac,avgccg ac:cagtgaa
gga,agaL,c acaatttccc cgacga,vc ,aaaaacag, cag,a,c,gc
aga,gaac,c cc,gaagac: aagacaccg ccg,g,ac,a L,g,g,gaga aact tcggcaach QN'OO cc ,gng,gca, av,gggg,ca
gggcacac:g gtgaccgtg: ccagc
Amino Acid Sequence of Humanized mAb2 Heavy Chain t 7 (h-
mAb2 VH-7) (SEQ ID NO:48):
flVQLVflSGGG LVQPGGSLR; SCAASGFTFS TYAMNWVRQA PGKGLEWVAR
IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSHKT EDTAVYYCVR
HGNFGNSYVS WFAYWGQGT; VTVSS
Polynucleotide Sequence Encoding Humanized mAb2 Variable Heavy Chain
Variant 7 (h-mAb2 VH-7) (SEQ ID NO:49):
gaggtgcagc ,gg,ggaaag cggcggagga ctggtgcagc caggtggcag
cctgcgac,g ,c Vgcgccg ,anggc,L cacc,,Lch aca,acgcca
tgaactgggt ggc: ctggaaagg ggc,ggag,g gg,ggccagg
atcaggtcca ag:acaacaa :atgcaacc gccg ac:cagtgaa
gga,agaL,c acaatttccc cgacga,vc ,aaaaacag, cag,a,c,gc
aga,gaac,c cc,gaagac: aagacaccg ccg,g,ac,a L,g,g,gaga cacggaaact tcggcaach Ok—Qk—QN'OO ,achgvcc ,gng,gca, av,gggg,ca
gggcacac:g gtgaccgtg: ccagc
Amino Acid ce of Humanized mAb2 Heavy Chain Variant 8 (h-
mAb2 VH-8) (SEQ ID NO:50):
flVQLVflSGGG LVQPGGSLR; FTFN TYAMNWVRQA PGKGLEWVAR
IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSHKT EDTAVYYCVR
SYVS WFAYWGQGT; VTVSS
Polynucleotide Sequence Encoding Humanized mAb2 Variable Heavy Chain
Variant 8 (h-mAb2 VH-8) (SEQ ID NO:51):
gaggtgcagc ,gg,ggach nggggaggc ttggtccagc ctggagggtc
cctgagach vcc,ngcag cctctggatt cacc:tcaac aca,acgc,a
tgaattgggt ccgccaggct ccagggaagg ggctggagtg ggttgcaagg
atcaggtcca agtacaacaa ,aLgcaacc eac,aegccg ac:ctgtgaa
gga,agaL,c accatctcaa attc aaagaac,ca ceg,a,chc
aaa:gaacag cotgaaaacc gaggacacgg ccg,g,a,,a c,g,g,gaga
cacggtaact tcggcaa,Lc ,,achg,cL ,ggeL,gceL ae,ggggaca
ggggacac,g ngach,gL c,Lcc
Amino Acid Sequence of Humanized mAb2 Variable Heavy Chain Variant
QV (h-mAb2 VL-QV) (SEQ ID NO:52):
flVQLVflSGGG LVQPKGSLK; SCAASGFTFN TYAMNWVRQA PGKGLEWVA?
IRSKYNNYAT YYADSVKDRF TISRDDSQS" LYLQMNNHKT EDTAMYYCV?
HGNFGNSYVS WFAYWGQGT; VTVSA
Polynucleotide Sequence Encoding Humanized mAb2 Variable Heavy Chain
Variant QV (h-mAb2 VL-QV) (SEQ ID NO:53):
gaggtgcagc ,gg,ggaaag cggcggagga ctggtgcagc caaagggatc
actgaaac,g ,cc,gcgccg gc:t cacc,,Laac aca,acgc,a
tgaattgggt gcgacaggca cotggcaagg gcc,ggag,g gg,ggcaagg
atcaggtcca agtacaacaa e,aLgcaacc gccg ac:ctgtgaa
gga,agaL,c acaatcagtc gcgacga,,c ca,e c,g,a,chc
agatgaacaa ,c,gaaaac: gaagacaccg cca,g,ac,a L,g,g,gcgg
cacggtaact :cggcaa,,c ,cL ,ggeL,gc,, ae,ggggaca
ggggacac,g ngach,ge c,Lcc
mAb2 Binds to Both Human and Cynomolgus Monkey CD3
] As discussed above, the mAb2 antibody was originally isolated based upon
its y to bind human CD3. In order to assess the y of mAb2 to bind to non-
human CD3, a capture ELISA was performed. Plates were coated with l ug/ml of
CD3 (either human or cynomolgus ) and incubated in the presence of various
concentrations of a ic variant of mAb2 dy (ch-mAb2) (containing the
variable sequences of mAb2 and the constant regions of a human antibody). As a
control, plates were also incubated with an antibody composed of the light chain of a
humanized mAb2 antibody and the heavy chain of the chimeric antibody. The results
of this experiment are shown in Figures 2A and 2B, and reveal that the chimeric
mAb2 variant exhibited equivalent binding to human CD3 and to cynomolgus
monkey CD3.
formed and their binding assessed using a capture ELISA. Plates were coated with l
”(T/m] nf this thrm‘pllnlm‘ r‘lnmnin nf 1111an fin? filp kph? nr “thn2”\ nnr‘l
Example 5
Analysis of g Characteristics of Variants of h-mAb2
Light and Heavy Chains
An analysis was conducted to determine the effect of variations in the
framework residues of the light chain of mAb2. Table 2 indicates the substitutions
studied.
Table 2
42 43 SEQ IDN
h-mAb2VL-1 F Q G Q A P R T 16
—_ 18
h—mAb2 VL-3 E - 20
h_mAb2 VL-4 22
—_ 24
—_ 26
h—mAb2 VL-7 E - 28
h_mAb2 VL-8 V E 30
—_ 32
Y V 7
A 36
- 4°
V 42
— 44
hVH-6M 72
hVH-7 A V 48
hVH-8L N A E V 55
hVH-8M N A N 74
Antibodies having mAb2 light chains of SEQ ID NO:11, but containing a
(Kabat numbered) tution of D4lG, H42Q, L43A, F44P, T45R, or G46T and
heavy chains of chimeric mAb2 (CDRs of mAb2 with hFRl-mFR2-hFR3-4) were
formed and their binding assessed using a capture ELISA. Plates were coated with l
ug/ml of the extracellular domain of human CD3 (soluble hCD3 or “shCD3”) and
Vdfl'dIlI VH-OIVI) are p'dITlCUl'dle preIerreu I01" pTOLIUCng 'dIlIlDOLllCS Il'dVC 'd IOWCI"
affinity for CD3 than antibodies composed of hVH-l, hVH-2, hVH-3, hVH-4, hVH-S,
ted in the presence of various concentrations of dy. The results (Figure
3) indicate that a substitution of T at Kabat position 46 eliminated the ability of the
antibody to bind to shCD3.
Additional studies were conducted to assess the impact of variations at Kabat
light chain positions 36, 38, 44 and 46. Antibodies were formed having an h-mAb2
VL-8, h-mAb2 VL-9 or h-mAb2 VL-lO light chain and the heavy chain of the mAb2
chimeric antibody and evaluated using the above-described capture ELISA. The
results of this experiment are shown in Figure 4, and reveal that the binding to shCD3
by an dy having the hVL-8 light chain was similar to that of an antibody having
the chimeric mAb2 light chain.
] Antibodies were also formed having an h-mAb2 VL-6, h-mAb2 VL-7 or h-
mAb2 VL-8 light chain and the heavy chain of the mAb2 chimeric antibody and
evaluated using the above-described capture ELISA (except that the plates were
coated with 0.5 ug/ml of shCD3 in phosphate buffered saline) to determine the impact
of additional substitutions at positions 36, 38 and 46. The results of this experiment
are shown in Figure 5, and reveal that the Kabat substitutions F36V and T46G were
sufficient to yield an antibody whose binding to shCD3 was similar to that of an
antibody having the chimeric mAb2 light chain.
The impact of substitutions in the sequence of the heavy chain of mAb2 was
ed by forming dies having the light chain of the chimeric mAb2 dy
and heavy chain h-mAb2 VH-S, h-mAb2 VH-6 or h-mAb2 VH-7 and ting
binding using the above-described capture ELISA (using a l ug/ml coating of
shCD3). The results of these investigations are shown in Figure 6. Antibodies were
additionally formed having the light chain of the chimeric mAb2 antibody and a
humanized variant of heavy chain h-mAb2 VH-4, h-mAb2 VH-7 or h-mAb2 VH-9.
Such antibodies were evaluated for binding using the described capture ELISA.
The results of these investigations are shown in Figure 7.
Heavy chains hVH-6L (and its t, hVH-6M), and hVH-8L (and its
variant VH-8M) are particularly preferred for producing antibodies have a lower
affinity for CD3 than antibodies composed of hVH-l, hVH-2, hVH-3, hVH-4, hVH-S,
Amino acid sequence of hVH-8M (SEQ ID NO:74):
«.VOT.V«.SGGG LVOPGGSLRL SCAASGFTFN TYAMNWVROA PGKGLEWVAR
hVH-6, hVH-7 or hVH-8 of Table 2. Such reduced affinity antibodies will preferably
be composed of either heavy chain hVH-6L or heavy chain VH-8L in combination
with any of light chain h-mAb2 VL-l, h-mAb2 VL-2, h-mAb2 VL-3, h-mAb2 VL-4,
h-mAb2 VL-S, h-mAb2 VL-6, h-mAb2 VL-7, h-mAb2 VL-8, h-mAb2 VL-9, or h-
mAb2 VL-lO. A particularly preferred deimmunized antibody will be composed of
heavy chain hVH-6L (or its variant, hVH-6M )and light chain h-mAb2 VL-6, or
heavy chain hVH-8L (or its variant, hVH-8M) and light chain h-mAb2 VL-6. The
sequences of such polypeptides are presented below:
Amino acid sequence of hVH-6L (SEQ ID NO:54):
flVQLVflSGGG LVQPGGSHRH SCAASGFTFN VQQA PGKGLEWVG?
:RNKY NYAT EYADSVKDQF TISRDDSKNS LYLQMNSHKT EDTAVYYCV?
{GNFG SYVS WFAYWGQGT; VTVSS
Amino acid sequence of hVH-8L (SEQ ID NO:55):
SGGG LVQPGGSHRH SCAASGFTFN TYAMNWVQQA PGKGLEWVA?
:RNKY NYAT EYADSVKDRF TISRDDSKNS LYLQMNSHKT YCV?
{GNFG SYVS WFAYWGQGT; VTVSS
Heavy chains hVH-6L and hVH-8L were further modified to produce
variants possessing an asparagine at position 52a (S52aN) modification. The amino
acid sequences and corresponding polynucleotide-encoding sequences of these
modified heavy chains are as follows:
Amino acid sequence of hVH-6M (SEQ ID NO:72):
flVQLVflSGGG LVQPGGSHRH FTFN TYAMNWVRQA PGKGLEWVG?
IRSKY NYAT EYAASVKDQF TISRDDSKNS LYLQMNSHKT YCV?
{GNFG SYVS WFAYWGQGT; VTVSS
Polynucleotide Sequence Encoding hVH-6M Variable Heavy Chain (SEQ
ID NO:73):
gaggtgcagc ,gg,ggach aggc ttggtccagc ctggagggtc
ac,c scc,ngcag chc,gga,L cacc,,caac gc,a
tgaattgggt ccgccaggct aagg ggc,ggag,g gg,,ggaagg
atcaggtcca agtacaacaa s,aLgcaacc gag,a,gccg ac:ctgtgaa
gga,agaL,c accatctcaa a,,c aaagaac,ca csg,a,chc
aaatgaacag cotgaaaacc gaggacacgg ccg,g,a,,a c,g,g,gaga
cacggtaact a,Lc ,achg,cL ngsL,gc,L as,ggggaca
ggggacac,g ngach,gL c
Amino acid sequence of hVH-8M (SEQ ID NO:74):
«.VQLVnSGGG LVQPGGSLRL SCAASGFTFN TYAMNWVRQA PGKGLEWVAR
9M di-l and light chain h-mAb2 VL-6, or heavy chain hVH-8L di-2 and light chain h-
mAb2 VL-6.
NYAT EYAASVKDRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR
HGNFGNSYVS WFAYWGQGTL VTVSS
Polynucleotide ce Encoding hVH-8M Variable Heavy Chain (SEQ
ID NO:75):
gagg:gcagc ,gg,ggach ,gggggaggc ttgg':ccagc ctggaggg:c
cctgagac,c occ,ngcag chc,gga,L cacc,,caac aca,acgc,a
tgaa:tgggt ccgccaggct ccagggaagg ggc,ggag,g ggwgcaagg
atcaggaaca agtacaacaa ,aLgcaacc gag,a,gccg ac:ctgtgaa
gga,agaL,c accatctcaa gaga,ga,oc aaagaac,ca cog,a,chc
aaa:gaacag cotgaaaacc gaggacacgg ccg,g,a,,a c,g,g,gaga
cacggtaact tcggcaayuc ,achgfiL ngoL,gc,L awggggaca
ggggacac,g gL c oLcc
Heavy chains hVH-8 di-l and hVH-8 di-2 are ularly preferred for
producing antibodies that are less immunogenic than dies composed of hVH-l,
hVH-2, hVH-3, hVH-4, hVH-S, hVH-6, hVH7 or hVH-8 of Table 2. Such
deimmunized antibodies will preferably be composed of either heavy chain hVH-8 di-
1 or heavy chain hVH-8 di-2 in combination with any of light chain h-mAb2 VL-l, h-
mAb2 VL-2, h-mAb2 VL-3, h-mAb2 VL-4, h-mAb2 VL-S, h-mAb2 VL-6, h-mAb2
VL-7, h-mAb2 VL-8, h-mAb2 VL-9, or h-mAb2 VL-lO. A particularly preferred
deimmunized antibody will be composed of heavy chain hVH-8 di-l and light chain
h-mAb2 VL-6, or heavy chain hVH-8 di-2 and light chain h-mAb2 VL-6.
Amino acid ce of hXR32VH-8 di-l (SEQ ID NO:56):
flVQLVflSGGG LVQPGGSLRL SCAASGFTFN VRQA PGKGLEWVA?
TRSKA SYTT YYAASVKGRF TISRDDSKNS LYLQMNSHKT ?DTAVYYCAR
{GNFG SYVS WFAYWGQGT; VTVSS
Amino acid sequence of hXR32VH-8 di-2 (SEQ ID NO:57):
flVQLVflSGGG LVQPGGSLRL SCAASGFTFN TYAMNWVRQA PGKGLEWVG?
TRSKA SYTT YYAASVKGRF TISRDDSKNS LYLQMNSHKT ?DTAVYYCAR
{GNFG SYVS QGT; VTVSS
Such deimmunized antibodies will preferably be composed of either heavy
chain hVH-8L di-l or heavy chain VH-8L di-2 in combination with any of light chain
h-mAb2 VL-l, h-mAb2 VL-2, h-mAb2 VL-3, h-mAb2 VL-4, h-mAb2 VL-S, h-mAb2
VL-6, h-mAb2 VL-7, h-mAb2 VL-8, h-mAb2 VL-9, or h-mAb2 VL-lO. A
particularly preferred deimmunized antibody will be composed of heavy chain hVH-
9M di-l and light chain h-mAb2 VL-6, or heavy chain hVH-8L di-2 and light chain h-
mAb2 VL-6.
EGNFGNSYVS WFAYWGQGTL VTVSA
Additional humanized variants of the mAb2 murine monoclonal antibody
variable heavy chain (SEQ ID NO:7) were also produced. The amino acid sequences
of such variants are presented below, with changes from SEQ ID NO:7 indicated in
boldface and underlining:
Amino Acid Sequence of variant “a” (151T Y52cA) of humanized mAb2
murine monoclonal antibody le heavy chain (SEQ ID NO:76):
flVKLLflSGGG LVQPKGSHKH SCAASGFTFN TYAMNWVQQA PGKGLEWVA?
EASKA.NYAT YYADSVKDRF TISRDDSQS" LYLQMNNHKT EDTAMYYCVR
{GNFE SYVS QGT; VTVSA
Amino Acid ce of variant “b” (ISlT N54S) of humanized mAb2
murine monoclonal antibody variable heavy chain (SEQ ID NO:77):
flVKLLflSGGG LVQPKGSHKH SCAASGFTFN TYAMNWVQQA PGKGLEWVA?
ERSKY §YAT YYADSVKDRF TISRDDSQS" LYLQMNNHKT EDTAMYYCV?
{GNFG SYVS WFAYWGQGT; VTVSA
Amino Acid Sequence of variant “c” (151T A56T) of humanized mAb2
murine monoclonal antibody variable heavy chain (SEQ ID NO:78):
flVKLLflSGGG LVQPKGSHKH SCAASGFTFN TYAMNWVQQA PGKGLEWVA?
ERSKY NYET YYADSVKDRF SQS" LYLQMNNHKT EDTAMYYCVR
{GNFG SYVS WFAYWGQGT; VTVSA
Amino Acid Sequence of variant “d” (151T Y52cA N54S) of humanized
mAb2 murine monoclonal antibody variable heavy chain (SEQ ID NO:79):
flVKLLflSGGG LVQPKGSHKH SCAASGFTFN TYAMNWVQQA PGKGLEWVA?
ERSK§.§YAT YYADSVKDRF TISRDDSQS" NHKT EDTAMYYCV?
{GNFG SYVS WFAYWGQGT; VTVSA
Amino Acid Sequence of variant “e” (151T N54S A56T) of humanized
mAb2 murine monoclonal antibody variable heavy chain (SEQ ID NO:80):
flVKLLflSGGG SHKH SCAASGFTFN TYAMNWVRQA PGKGLEWVA?
ERSKY §YET KDRF TISRDDSQS" LYLQMNNHKT YCV?
{GNFG SYVS WFAYWGQGT; VTVSA
] Amino Acid Sequence of variant “f” (151T Y52cA N54S A56T) of
humanized mAb2 murine monoclonal antibody variable heavy chain (SEQ ID
flVKLLflSGGG LVQPKGSHKH SCAASGFTFN TYAMNWVQQA PGKGLEWVA?
ERSK§.§YET YYADSVKDRF TISRDDSQS" LYLQMNNHKT EDTAMYYCV?
{GNFG SYVS WFAYWGQGT; VTVSA
Amino Acid Sequence of variant “9” (151T D6lA) of humanized mAb2
murine monoclonal antibody variable heavy chain (SEQ ID NO:82):
SGGG LVQPKGSHKH SCAASGFTFN VQQA WVA?
ERSKY NYAT YYAESVKDRF TISRDDSQS" LYLQMNNHKT EDTAMYYCVR
{GNFG SYVS WFAYWGQGT; VTVSA
Amino Acid Sequence of t “h” (151T D65G) of humanized mAb2
murine monoclonal antibody variable heavy chain (SEQ ID NO:83):
flVKLLflSGGG LVQPKGSLK; SCAASGFTFN TYAMNWVQQA PGKGLEWVA?
ERSKY NYAT YYADSVKERF TISRDDSQS" LYLQMNNHKT EDTAMYYCV?
{GNFG SYVS WFAYWGQGT; VTVSA
Amino Acid Sequence of variant “i” (ISlT Y52cA N54S D6lA) of
humanized mAb2 murine monoclonal antibody variable heavy chain (SEQ ID
NO:84):
nVKLLnSGGG LVQPKGsnKn SCAASGFTFN TYAMNWVRQA WVAR
ERSK§_§YAT YYAgSVKJRF TISRDDSQS" LYLQMNNnKT ?DTAMYYCVR
{GNFG syvs WFAYWGQGT; VTVSA
Amino Acid Sequence of variant “j” (ISlT Y52cA N54S D65G) of
humanized mAb2 murine monoclonal antibody variable heavy chain (SEQ ID
NO:85):
SGGG LVQPKGSLK; SCAASGFTFN TYAMNWVRQA PGKGLEWVAR
ERSKA.§YAT YYADSVKERF TISRDDSQS" NHKT EDTAMYYCV?
{GNFE SYVS WFAYWGQGT; VTVSA
] Amino Acid Sequence of variant “k” (ISlT Y52cA N54S D6lA D65G) of
humanized mAb2 murine monoclonal antibody variable heavy chain (SEQ ID
NO:86):
nVKLLnSGGG LVQPKGSLK; SCAASGFTFN TYAMNWVRQA PGKGLEWVAR
ERSK§_§YAT YYAgSVKgRF TISRDDSQS" LYLQMNNnKT ?DTAMYYCVR
{GNFG syvs WFAYWGQGT; VTVSA
Amino Acid Sequence of variant “2k” (ISlT Y52cA N54S D6lA D65G
(VH8-A49G V93A)) of humanized mAb2 murine monoclonal antibody variable
heavy chain (SEQ ID NO:87):
nVQLVnSGGG LVQPGGSLR; FTFE VRQA PGKGLEWVGR
ERSK§_§YTT YYAgSVKgaF TISRDDSKNS LYLQMNsnKT YCAR
{GNFG syvs WFAYWGQGT; VTVSS
ALVA; AAWAAAWAA way, ‘4 v “‘4‘va vvwv I’VLAVLAAAvw. A vavv nvxv vvwvvw "AVA; A r4101 ALL;
of the extracellular domain of CD3 (soluble CD3) (either human or cynomolgus
Amino Acid Sequence of variant “5k” (151T Y52cA N54S D6lA D65G
(VH8-V93A)) of zed mAb2 murine monoclonal antibody variable heavy chain
(SEQ ID NO:88):
+.VQT.V«.SGGG LVQPGGSLR; SCAASGFTFE VRQA PGKGLEWVER
gRSKg §YTT YYAgSVKgaF TISRDDSKNS T.YT.QMNS'.KT '«TDTAVYYCAR
{GNFG syvs WFAYWGQGT; VTVSS
All such additional humanized variants of the mAb2 murine monoclonal
antibody variable heavy chain may be employed to form the deimmunized dies
of the present invention. Such additional deimmunized and humanized antibodies
will preferably be composed of any of heavy chains: a, b, c, d, e, f, g, h, i, j, k, 2k or
5k, in ation with any of light chain: h-mAb2 VL-l, h-mAb2 VL-2, h-mAb2
VL-3, h-mAb2 VL-4, h-mAb2 VL-S, h-mAb2 VL-6, h-mAb2 VL-7, h-mAb2 VL-8,
h-mAb2 VL-9, or h-mAb2 VL-lO. A particularly preferred deimmunized antibody
will be composed of heavy chain 2k or 5k and light chain h-mAb2 VL-6, or heavy
chain hVH-8M8L di-2 and light chain h-mAb2 VL-6. Variants 2k and 5k bind to
Protein A in the variable region, thus facilitating the purification of molecules (such
as diabodies) that may lack Fc regions or other domains that may be used to sequester
such molecules from other molecules. ts hVH-8M, hVH-8L. hVH-6M and
hVH-6L exhibit reduced immunogenicity relative to their respective parental
polypeptides.
The invention particularly ns nized and humanized antibodies
composed of heavy chain hVH-8 and light chain VL-6. The invention additionally
particularly concerns deimmunized and humanized antibodies composed of heavy
chain hVH-4 and light chain VL-6. The ion additionally particularly concerns
deimmunized and humanized dies composed of heavy chain hVH-2k and light
chain VL-6
Example 6
Analysis of Binding teristics of Variants of Chimeric and Humanized
mAb2 Light and Heavy Chains
In order to assess the ability of chimeric and humanized mAb2 to bind to
non-human CD3, a capture ELISA was performed. Plates were coated with l ug/ml
of the extracellular domain of CD3 (soluble CD3) (either human or cynomolgus
receptor (EGFR) M diabody “ERBITUXTM-h-mAbZ”).
monkey) and incubated in the presence of various concentrations of antibody. The
results of this experiment are shown in s 8A and 8B, and reveal that mAb2 and
its humanized variant exhibited equivalent g to soluble human CD3 and to
soluble cynomolgus monkey CD3.
Example 7
Quantitation of g of mAbZ to Human and Cynomolgus Monkey CD3
In order to quantitate the extent of binding between mAb2 and human or
cynomolgus monkey CD3, BIACORETM analyses were conducted. BIACORETM
es measure the dissociation off-rate, kd. The binding affinity (KD) between an
antibody and its target is a fimction of the kinetic constants for association (on rate,
ka) and dissociation (off-rate, kd ) according to KD= kd/ka. The BIACORETM is
uses surface plasmon resonance to directly measure these kinetic ters. Anti-
CD3 antibody mAb2 (6.3-100 nM) was immobilized to a support using anti-EK
antibodies and incubated in the presence of soluble human CD3 (shCD3) or soluble
cynomolgus monkey CD3 (scCD3). The time course of dissociation was measured
and a bivalent fit of the data conducted. The results of the BIACORETM es are
shown in s 9A-9D. The kinetic data is summarized in Table 3.
Table 3
Antibod ka kd
ch-mAb2 1.7 x 10 M" sec" 2.5 x 10" sec"
-1 -1 -3 -1
n10 a d
ch-mAb2 1.6 x 10 M" sec" 2.3 x 10" sec"
h-mAb2 1.7 x 10 M" sec" 4.1 x 10" sec"
Example 8
Bispecific Binding Data for DARTTM Diabodies Containing CDRs of h-mAb2
The CDRs of humanized mAb2 2) were used to produce a series of
DARTTM diabodies having an anti-CD3 first epitope binding site and a second epitope
binding site capable of binding to Her2/neu (DARTTM diabody h-mAb2”), or
to CD19 (DARTTM diabody “CD19-h-mAb2”) or to the epidermal grth factor
receptor (EGFR) (DARTTM diabody “ERBITUXTM-h-mAbZ”).
/ \ \ /
QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TS Q KPGQAPRGLT
GGTNKRAPWT SLLG GKAALTT TGA OAfl D-I.ADYYC ALWYSNITWVF
eu-h-mAb2 DARTTM Diabody
Amino acid sequence of the hXR32VL-Her—2VH E coil of the Her2-h-mAb2
DARTTM diabody (the linkers between the hXR32VL sequence and the Her2VH
sequence and between the Her2VH ce and the E coil ce are underlined)
(SEQ ID NO:58):
QAVVTQITPSL TVSPGGTVTL TCRSSTGAVT TS YANWVQQ KPGQAPRGLT
GGTNKRAPWT PAQFSGSH G GKAALTT TGA QA*I D.ADYYC ALWYSNITWVF
GGGTKITTVLG GGGSGGGGQV QLQQSGPT IV KPGASLKLSC TASGFNT KDT
YT HWVKQKP. QGIITIW G? Y PTNGYTRY DP KFQ D<ATT TA DTSSNTAYI4Q
VSRLTST4 DTA VYYCSRWGG D GFYAMDYWGQ GASVTVSSGG CGGGKVAATK
TKVAAI I<TKV AAI<ITKVAAII KT
Amino acid sequence of the Her2VL-hXR32VH-K coil of the Her2-h-mAb2
DARTTM diabody (the linkers between the Her2VL sequence and the hXR32VH
sequence and between the hXR32VH sequence and the K coil sequence are
underlined) (SEQ ID NO:59):
DTV TQS {KF MSTSVGDRVS TTCKASQDVN TAVAWYQQKP GHSPKLLTYS
ASFQYTGVP D RFTGNRSGT D SVQA ADLAVYYCQQ HYTTPPTFGG
GTKIIT. KRAG GGSGGGGTVQ LVTSGGGHVQ PGGSI I? ISCA ASGFTFNTYA
MNWVRQAPG < GLTWVA? RS KY NYATYYA DSVK jQFTT S RDDSKNSLYL
QMNS I<TTDT AVYYCVRTG FG SYVSWFA TVTV SSGGCGGGTVI
AATI*.<*.VAAH TKTVAAI ITKT VAALTK
CD19-h-mAb2 DARTTM y
Amino acid sequence of the CDl9VL-hXR32VH-E coil of the CDl9-h-
mAbZ DARTTM diabody (the linkers between the CDl9VL sequence and the
hXR32VH sequence and between the hXR32VH sequence and the E coil sequence
are underlined) (SEQ ID NO:60):
QSPAS QRAT TSCKASQSVD YDGDSYL WY QQTPGQPPKT
IITYDAS IVS GTPPRFSGSG SGTDhTLN { PVTKVDAATY ITDPW
TFGGGT<U*I {GGGSGGGGIT VQLVTSGGGU VQPGGSIKIS CAASGFTFNT
YAM WVRQAP GKGTITWVAR RSKY NYATY YADSVKDRFT TSRDDSKNST
YLQ NSII<TIT DTAVYYCVRT G FG SYVSW FAYWGQGTTV TVSSGGCGGG
TVAALTKTVA ALTKTVAAHT KflVAALflK
Amino acid sequence of the hXR32VL-CD19VH-K coil of the CDl9-h-
mAbZ DARTTM diabody (the linkers between the hXR32VL ce and the
CDl9VH sequence and between the CDl9VH sequence and the K coil sequence are
ined) (SEQ ID NO:61):
QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TS YANWVQQ KPGQAPRGLT
APWT PARFSGSLLG GKAALTT TGA QA dlD-I.ADYYC ALWYSNITWVF
GGGT<LTVLG GGGSGGGGQV AELV RPGSSVKISC KASGYAFSSY
QQPG QGTIflW GQ W PGDGDTNYNG KFKGKATLTA DESSSTAYMQ
LSSLAS_EDSA VYFCARQETT YAMD YWGQGTTVTV GGKV
AATIKTKVAATI KTKVAAI<TK VAAIKT
ERBITUXTM-h-mAb2 DARTTM Diabody
Amino acid sequence of the hXR32VL-EGFRVH-E coil of the
ERBITUXTM-h-mAbZ DARTTM diabody (the linkers between the hXR32VL
sequence and the EGFRVH sequence and between the EGFRVH sequence and the E
coil sequence are underlined) (SEQ ID NO:62):
QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TS YANWVQQ KPGQAPRGLL
GGTNKRAPWT PAQFSGSLLG GKAALT: TGA QATIDIADYYC ALWYSNLWVF
GGGTKLTVLG GGGSGGGGQV QL<QSGPGLV QPSQSLSL TC TVSGFSLT Y
GVHWVQQSPG KGHflWTGV W SGGNTDYNTP NKD FF<M
SLQS DTA__ YYCARALTYY DYEFAYWGQG TLVTVSSGGE EGGflVAALnK
IVAATflKflVA AALn K
Amino acid sequence of the EGFRVL-hXR32VH-K coil of the
ERBITUXTM-h-mAbZ DARTTM diabody (the linkers between the EGFRVL sequence
and the hXR32VH sequence and between the hXR32VH sequence and the K coil
sequence are underlined) (SEQ ID NO:63):
D"H'ITQSPV" HSVSPGERVS FSCRASQS G TN {WYQQRT NGSPRHL"KY
ASflS SG PS KESGSGSGTD hTTIS NSVflS *ID ADYYCQQ NNNWPTTFGA
GT<'IT IKGGG VQLV TSGGGTIVQPG SCAAS GFTFNTYAMN
WVRQAPGKGH flWVAK RSKY NYATYYADS VKDRFTLSQD DSKNSLYLQM
NSH<TTDTAV YYCVRTGNFG AYW GQGTJVTVSS GGCGGGKVAA
LKT<VAALKT <VAAH<TKVA ALK_3
Such DARTTM diabodies were found to be capable of binding to cynoniolgus
monkey CD3 (Figures 10A—10C).
The CDRs of humanized mAb2 (h-mAb2) were used to produce a fiarther
series of DARTTM diabodies having an anti-CD3 first epitope binding site and a
second e binding site capable of binding to B7-H3 (DARTTM diabody “B7-H3-
b2” and B7-H3h-n1Ab2”).
B7-H3h-mAb2 DARTTM Diabody
Amino acid sequence of the hBRCA69DVL-hXR32VH-E coil of the B7-H3-
l-h-mAb2 DARTTM diabody (the linkers between the hBRCA69DVL sequence and
LJL‘ULLLHJHHLLL y LLVvaxvLuLL‘ L.4_4_\_; ULHLHL w vaLL y VKJKJ \vavvv
T.KITKVAALKT KVAALKEKVA ALKE
the hXR32VH sequence and between the hXR32VH sequence and the E coil
sequence are underlined) (SEQ ID NO:64):
DT Q TQSPSS LSASVG DRVT TTCRASQDTS YLNWYQQKP F .TYY
GVPSJ RFSGSGSGTD FTLTT SSUQP LD ATYYCQQ GNTLPPTFGG
GT (UL <GGG TVQL VTSGGGLVQP GGS.¥ .SCAA SGFTFNTYAM
NWVRQAPGKGI ASK Y AD SVK DRFT IISR DDSKNSLYLQ
MNSH (TTDTA VYYCV {G F G SYVSWFAY WGQGTLVTVS SGGCGGIGLVA
AL L<LVAAL. <. *KLV AAT. -—uLK
Amino acid sequence of the hXR32VL-hBRCA69DVH-K coil of the B7-
H3-l-h-n1Ab2 DARTTM diabody (the linkers between the hXR32VL sequence and the
hBRCA69DVH sequence and between the hBRCA69DVH sequence and the K coil
sequence are underlined) (SEQ ID NO:65):
QAVVTQEPSL TVSPGGTVTL TCKSSTGAVT TS YANWVQQ KPGQAPRGLT
APWT RFSGSLLG G<AAI .TIITGA QA LD LADYYC ALWYSNILWVF
GGGTKLTVLG QILVQSGAILVK KPGASVKVSC FTSY
WMQWVQQAPG PGDG DTRYTQ KF I TA DKSTSTAYMI*_‘4
USSTRSITDTA R .WYFDVWGQ GTTVTVSSGG CGGGKVAAL <
TKVAAI. (TKV KI_‘_.
B7-H3h-mAb2 DARTTM Diabody
Amino acid sequence of the hBRCA84DVL—hXR32VH-E coil of the B7-H3-
2-h-n1Ab2 DARTTM diabody (the linkers between the hBRCA84DVL sequence and
the hXR32VH sequence and between the hXR32VH ce and the E coil
ce are ined) (SEQ ID NO:66):
DL Q' .TQSPSF LSASVG DRVT TTCKASQNV D TNVAWYQQKP I .TYS
ASYQYSGVPS RFSGSGSGTD FTTTT SSTQP TDFATYYCQQ YNNYPFTFGQ
GT <I.L {GGG GSGGGGITVQI. VITSGGGTVQP GGSI.¥ .SCAA SGFTFNTYAM
NWVRQAPGKG ImWVA? QS< Y NYATYYA D SVK DRFT IISR DDSKNSLYLQ
TTDTA VYYCVK {G F G SYVSWFAY WGQGTLVTVS SGGCGGGLVA
AL L<LVAALL (LVAAH *KLV AAT.TK
Amino acid sequence of the hXR32VL-hBRCA84DVH-K coil of the B7-
H3h-n1Ab2 DARTTM diabody (the linkers between the hXR32VL sequence and the
hBRCA84DVH sequence and between the hBRCA84DVH sequence and the K coil
sequence are underlined) (SEQ ID NO:67):
QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TS YANWVQQ KPGQAPRGHT
GGTNKKAPWT PAQFSGSH .G G<AALTII TGA QA LD LA DYYC ALWYSNILWVF
JTVLG GGGSGGGGTV QI.VITSGGGTIV QPGGSLQLSC AASGFTFSSF
GM {WVQQAPG KGI: LWVAY S SDSSAIIYYAD TV {GAFTT SR DNAKNSI .Y .Q
MNSLRDIL DTA VYYCGRGKLN YYGSRLDYW GQGTTVTVSS GGCGGG<VAA
LKITKVAAI .KT KVAALKIL<VA AI .KT
PGLICIILD lclllalll uuucr LIIC balC UL L11C11 PCIDUIIGI Pllyblblallb uuuug LIIC qulDC UL LIIC
study.
Such DARTTM diabodies were found to be e of binding to soluble
cynomolgus monkey CD3 (Figure 10D).
Dual Affinity Retargeting Reagents (DARTTMs) Diabodies Specific For HER2/neu and
CD3 Mediate Potent Redirected T-Cell Killing
Dual affinity retargeting reagent (DARTTM) diabodies c for HER2/neu
and CD3 are prepared. Such DARTTM diabodies have the ability to localize a T-cell
(by binding such T-cell to the nding portion of a CD3-binding DARTTM
diabody) to the on of a tumor cell (by binding such cancer cell to the HER2/neu-
binding portion of the DARTTM diabody). The localized T-cell can then mediate the
killing of the tumor cell in a process termed herein “redirected” killing.
The dual affinity eting reagent (DARTTM) diabody specific for
HER2/neu and CD3 is constructed having the anti-HER2/neu variable domains of
trastuzumab and anti-CD3 variable domains of h-mab2 VH-8 and h-mab2 VL-6 (SEQ
ID NOS: 58-59).
In order to demonstrate the ability of DARTTM diabodies to mediate such
redirected killing of cancer cells, the above-described HER2/neu x CD3 DARTTM
diabody is incubated at various concentrations with target tumor cells (SKOV-3 tumor
cells, SKBR-3 tumor cells, A549 tumor cells, and MCF-7 tumor cells) and effector
resting PBMC (E:T ratio = 30:1) and cytotoxicity is determined (LDH Assay). The
results of these investigations demonstrate the ability of the HER2/neu x CD3
DARTTM diabody to mediate redirected killing of tumor cells.
Example 10
Anti- TCR Monoclonal dy y for Patients with Autoimmune
Diabetes
Patients: Forty patients with Type 1 diabetes are recruited for participation
according to the ing criteria: between 7 and 20 years of age, within 6 weeks of
diagnosis according to the American Diabetes Association criteria, and confirmation
of the presence of anti-GAD65, anti-ICAS 12, and/or anti-insulin autoantibodies. The
patients remain under the care of their al physicians during the course of the
study.
Annunnunnu ALL kuv u; LLULLLLWL wavvuv wVLvaLLVV. L wvaLqu “AV “Lvavvu uv anvv w LLULLLLWL
diet, and remain under the care of their personal physician throughout the duration of
] Eligible patients are randomly assigned to a control group and a humanized
anti- CD3 antibody ) (comprising h-mab2 VH-8 and h-mab2 VL-6) treatment
group. After randomization, blood samples are drawn to establish baseline HAlc
levels, a pretreatment ide response to a MMTT is established and a
pretreatment FPIR to IGTT is performed. Patients in both groups are hospitalized to
receive either a 6-day course treatment of the zed anti- CD3 monoclonal
antibody (N297Q) or placebo. The antibody is administered intravenously in the
following dosage: 17 ug/m2 on day 1, 34.3 ug/m2 on day 2, 69 ug/m2 on day 3, 137.6
ug/m2 on day 4, and 275.3 ug/m2 on days 5 and 6. Alternatively, antibody may be
administered intravenously in the following dosage: 1.6 ug/kg/day on day 1; 3.2
ug/kg/day on day 2; 6.5 ug/kg/day on day 3; 13 ug/kg/day on day 4; and 26
ug/kg/day on days 5 through 14. In dose escalation studies, the treatment may be,
e.g., 1.42 ug/kg/day on day l; 5.7 ug/kg/day on day 2; 11 ug/kg/day on day 3; 26
ug/kg/day on day 4; and 45.4 ug/kg/day on days 5 through 14. In subsequent studies,
the therapy is altered to increase dosage and/or decrease the time course of treatment.
For example, in subsequent studies patients may be stered a 4 day treatment:
6.4 ug/kg/day on day l; 13 ug/kg/day on day 2, and 26 ug/kg/day on days 3 and 4.;
during additional dose escalation studies, the treatment may be 8 ug/kg/day on day 1;
16 day on day 2; and 32 ug/kg/day on days 3 and 4.
During initial studies the antibody dosage on the first three days of treatment
is stered via slow infusion IV over 20 hours to r for e reactions.
Subsequent studies will decrease the time of administration and/or split the dosage
into 2 to 4 equal parts to be administered as bolus injections evenly distributed over
the course of 12 hours. Patients in the control group undergo metabolic and
immunologic tests but do not receive monoclonal dies. Patients are monitored
throughout the study for immunosuppressive effects of the anti- CD3 monoclonal
antibody (N297Q).
ts are monitored for 18 months after the treatment. B-cell fianction is
determined every 6 months in the case of ed glucose nce and every 12
months in case of normal glucose tolerance. Patients are allowed to have a normal
diet, and remain under the care of their personal physician throughout the duration of
the study. Immunological assays are repeated in intervals of 6 months. Insulin
therapy will be given to the patients as directed by their personal physician.
B-cell filnction will be analyzed according to the changes of the C-peptide
levels as measured by radioimmunoassay. After drawing samples for baseline C-
peptide and glucose, the patients are given a mixed meal. The C-peptide levels are
measured in samples drawn after 15, 30, 60, 90, 120, 150, 180, 210, and 240 min. The
C-peptide response to the mixed-meal tolerance test (MMTT) is expressed as the total
area under the response curve (AUC). A change in the se is considered to have
occurred if the response differs by more than 7.5 percent from the response at study
entry. The patients’ C-peptide ses to MMTT are continuously monitored 6
months, 9 months, 12 months, 15 months and 18 months after the ent.
Alternatively, the B-cell fianction is assessed by FPIR to IGTT. Serum insulin levels
are measured by a modification of a double-antibody radioimmunoassay method using
monoiodinated tyrosine Al4—labeled insulin ham Pharmacia). FPIR is
calculated as the sum of insulin levels at l and 3 minutes after a glucose load (0.5
g/kg). Glycosylated obin levels are measured by latex-agglutination inhibition
test.
Immunological Monitoring: The level of autoantibodies against GAD65,
A512, and insulin are measured with radiobinding assays as known in the art
(e.g., Woo et al., 2000, J. Immunol s 244:91-103). A and HLADQB
genotyping are performed by direct sequencing of exon 2 polymorphisims after
PCR amplification. The level of cytokines in serum after the administration of the
monoclonal antibody is ed by enzyme-linked sorbent assay (ELISA).
Production of anti-idotype dies is monitored by ELISA assay using a plate
bound anti-CD3 ) or by flow cytometry to measure blockade of binding of
anti-CD3-FITC to the CD3 chain of TCR.
Statistical Analysis: Data analysis will be conducted on residual beta-cell
function, autoantibody level, cytokine level, and glycosylated hemoglobin level. A x2
analysis will be performed to test the effect of drug treatment before and after drug
ULL U1 u Lu111u1 UULL \U)’ U111u1115 ouu11 Uu11uv1 UULL LU LLLU 1.1/11.) U111u1115 PU1 L1U11 U1
administration. Comparison between the control group and the treatment group will
be made with the Mann-Whitney U test.
Example 1 1
Dual Affinity Retargeting Reagents (DARTTMs) Diabodies Specific for B7H3 and CD3
Mediate Potent Redirected T-Cell Killing
Dual affinity retargeting reagents (DARTTM) diabodies specific for the B7H3
antigen and CD3 were prepared. B7H3 has been histologically detected in
tumor cell lines (Chapoval, A. et al. (2001) .‘ A Costimulatory Molecule For T
Cell Activation and IFN—y Production,” Nature Immunol. 274; Saatian, B. et al.
(2004) “Expression 0f Genes For B7-H3 And Other T Cell Ligands By Nasal
lial Cells During Difi’erentiation And Activation,” Amer. J. Physiol. Lung Cell.
Mol. Physiol. 287:L217—L225; Castriconi et al. (2004) “Identification 0f 4Ig-B7-H3
As A Neuroblastoma-Associated le That Exerts A tive Role From An NK
Cell-Mediated Lysis,” Proc. Natl. Acad. Sci. (USA) lOl(34):l2640-l2645); Sun, M.
et al. (2002) “Characterization of Mouse and Human B7—H3 Genes,” J. Immunol.
168:6294-6297). Several independent studies have shown that human malignant
tumor cells t a marked increase in expression of B7-H3 protein and that this
increased expression was associated with sed disease severity (Zang, X. et al.
(2007) “The B7 Family And Cancer Therapy: Costimulation And Coinhibition,” Clin.
Cancer Res. 13:5271-5279), suggesting that B7-H3 is exploited by tumors as an
immune evasion pathway (Hofmeyer, K. et al. (2008) “The Contrasting Role 0fB7—
H3,” Proc. Natl. Acad. Sci. (USA) 105(30):10277-10278).
The CD3 binding portion of such DARTTM diabodies was composed of the
above-described light and heavy variable regions of humanized anti-CD3 mAb2 (h-
mAb2 VH-8 and h-mAb2 VL-6). The B7H3 portion of such DARTTM diabodies was
composed of 4D-2 Light Chain and hBRCA84D-2 Heavy Chain (SEQ ID
NOS. 64-65).
Such DARTTM diabodies have the ability to localize a T-cell (by binding
such T-cell to the CD3-binding portion of a CD3-binding DARTTM diabody) to the
location of a tumor cell (by binding such cancer cell to the B7H3 binding portion of
lU‘I-
the DARTTM diabody). The localized T-cell can then mediate the killing of the tumor
cell Via the s of ected” killing.
In order to demonstrate the ability of such DARTTM diabodies to mediate
such redirected killing of cancer cells, the DARTTM diabody was incubated at various
concentrations with target tumor cells (A498 tumor cells, RECA905021E tumor cells)
and effector resting PBMC (E:T ratio = 30:1) and cytotoxicity was ined (LDH
Assay). A DARTTM y (4420-h-mAb2) haVing dual specificity for CD3 (h-
mAb2) and fluorescein (antibody 4420) was employed as a control.
4420-h-mAb2 DARTTM Diabody
Amino acid sequence of the 4420VL-hXR32VH-E coil of the 4420-h-mAb2
DARTTM diabody (the linkers between the 4420VL ce and the hXR32VH
sequence and between the hXR32VH sequence and the E coil sequence are
underlined) (SEQ ID NO:68):
DVVMTQTPFS LPVS_JGDQAS :SCRSSQSLV HSNGNTY_JRW YLQKPGQSPK
VLIYKVS RF SGVPDQFSGS GSGTDhTLK SRV*A*IDIGV THVP
WTFGGGT<Ifl KGGGSGGGG *IVQUVISGGG LVQPGGSIRU SCAASGFTFN
TYAMNWVRQA PGKGHIWVAR __RS<Y NYAT YYADSVKDRF TISRDDSKNS
LYLQMNSH<T YCVR {G FG SYVS WFAYWGQGTL VTVSSGGCGG
GfiVAALn<flV AALfl<flVAAH flKIVAALnK
Amino acid sequence of the hXR32VL-4420VH-K coil of the 4420-h-mAb2
DARTTM diabody (the s between the L sequence and the 4420VH
sequence and between the 4420VH sequence and the K coil sequence are underlined)
(SEQ ID NO:69):
QAVVTQ?PSH TVSPGGTVT; TCQSSTGAVT TS YANWVQQ KPGQAPRGH"
GGTNKRAPWT PARFSGSLLG GKAALT: TGA QA*IDflADYYC A'IWYSNHWVF
TVUG GGGSGGGG?V KTIDTTGGGTIV QPGQPM<LSC VASGFTFSDY
WMNWVRQSPfl KGHflWVAQ R NKPYNYETYY SDSV<GRFTI SRDDSKSSVY
TIQMNN IRV*ID MG YYCTGSY YG DYWGQGT SVTVSSGGCG GGKVAAIKTK
VAALKEKVAA LK?<VAAL<?
] The results of these investigations (Figures B) demonstrate the
ability of the B7H3 x CD3 DARTTM diabodies to mediate redirected killing of tumor
cells expressing B7H3.
RECA47VH sequence and between the RECA47VH sequence and the E coil
Example 12
Dual Affinity Retargeting Reagents (DARTTMs) Diabodies Specific For A33 and CD3
e Potent Redirected T-Cell Killing
Dual affinity retargeting ts (DARTTM) diabodies specific for the A33
antigen and CD3 (“A33-h-mAb2” DARTTM diabody) were prepared. A33 is a
membrane antigen that is expressed in normal human colonic and small bowel
epithelium and >95% of human colon cancers (Heath, J.K. et al. (1997) “The Human
A33 Antigen Is A Transmembrane Glycoprotez'n And A Novel Member Of The
Immunoglobulz’n Supely’amily,” Proc. Natl. Acad. Sci. (USA) 94:469-474).
Such DARTTM diabodies have the ability to localize a T-cell (by binding
such T-cell to the CD3-binding portion of a CD3-binding DARTTM diabody) to the
location of a tumor cell (by binding such cancer cell to the A33 binding portion of the
DARTTM diabody). The localized T-cell can then mediate the g of the tumor cell
Via the s of “redirected” g.
The CD3 binding n of such DARTTM diabodies was composed of the
described light and heavy variable regions of humanized mAb2 (h-mAb2 VH-8
and h-mAb2 VL-6). The A33 portion of such DARTTM diabodies was composed of
antibody RECA47.
A33-h-mAb2 DARTTM Diabody
Amino acid ce of the RECA47VL-hXR32VH-K coil of the A33-h-
mAb2 DARTTM diabody (the linkers between the VL sequence and the
hXR32VH sequence and n the hXR32VH sequence and the K coil sequence
are underlined) (SEQ ID NO:70):
Q"VHTQSPA" SASPGERVT TCSARSSIS FMYWYQQKPG "YDT
SNLASGVPV? FSGSGSGTSY SET SQMflAfl DAATYYCQQW SSYPLTFGSG
TKHEL<RGGG SGGGGEVQLV ESGGGUVQPG GSUQHSCAAS GFTFNTYAMN
WVRQAPGKGH flWVAR QSKY NYATYYADS VKDQFTISRD DSKNSLYLQM
NSH<TEDTAV YYCVRiGNFG SYVSWFAYW GQGTLVTVSS GGCGGGKVAA
LK?<VAALK? <VAAH<EKVA ALKE
Amino acid sequence of the hXR32VL-RECA47VH-E coil of the A33-h-
mAb2 DARTTM y (the linkers between the hXR32VL sequence and the
RECA47VH sequence and between the RECA47VH sequence and the E coil
sequence are underlined) (SEQ ID NO:71):
Molecular Pathogenesis l o New Y herapeutic Perspectives,” Haematologica 91 :1 1-16;
Jeon, H]. et al. (1998) “Establishment And Characterization Of A Mantle Cell
EPSL TVSPGGTVTL GAVT TS Q RGL:
APWT PARFSGSLLG GKAAHT"TGA QAflDflADYYC ALWYSNLWVF
GGGTKLTVLG GGGSGGGGQV QLQQS PELV KPGASVKISC KASGYTFSGS
WMNWV<QRPG QGHflW G? Y PGDGETNYNG KF<D<ATLTA DKSSTTAY E
LSSLTSVDSA IYG NVYFDVWGAG TTVTVSSEEE EggflVAALn<
flVAALflKflVA ALn<flVAALn K
In order to demonstrate the ability of such DARTTM diabodies to mediate
such redirected killing of cancer cells, the DARTTM diabody was incubated at various
concentrations with target tumor cells (Colo205 tumor cells, RECA905021E tumor
cells) and effector resting PBMC (E:T ratio = 30:1) and cytotoxicity was determined
(LDH Assay). The results of these investigations (Figures 12A-12E) trate the
ability of the A33 x CD3 DARTTM ies to mediate redirected killing of tumor
cells expressing A33.
Example 13
Dual Affinity Retargeting ts (DARTTMs) ies Specific For CD3
Cause Redirected T-Cell-Mediated Killing Equivalent to That of Other Human-Specific
CD3 Diabodies
In order to further assess the CD3-specific dual affinity retargeting reagents
(DARTTM) ies of the present invention, the capacity of the above-described
CD19-h-mAb2 DARTTM diabody to cause redirected T-cell-mediated killing was
compared to that of the CD19 x CD3 DART diabody of Moore, RA. et al. (2011)
(“Application OfDual y Retargeting Molecules To e Optimal Redirected
T-Cell g 0f B-Cell Lymphoma,” Blood 117(17):4542-4551). The CD19-h-
mAb2 DARTTM diabody exhibits specificity to human as well as non-human CD3; the
CD19 x CD3 DART diabody of Moore, RA. et al. (2011)) exhibits specificity only to
human CD3.
Accordingly, Raji human B-cell lymphoma cells (see, Drexler, H.G. et al.
(1998) “History And Classification Of Human Leukemia-Lymphoma Cell Lines,”
Leuk. Lymphoma 31(3-4):305-3l6; Amdt, R. (1984) “Demonstration 0f C3-Binding
Circulating Immune Complexes Using Raji, Conglutinin And Anti-C3 Assays--A
Critical Review,” Immun. Infekt. 12(1):3-11) or JeKo-l human mantle cell lymphoma
cells (Salaverria, I. et al. (2006) “Mantle Cell Lymphoma: From Pathology And
Molecular Pathogenesis To New Therapeutic Perspectives,” Haematologica 91 :1 1-16;
Jeon, H]. et al. (1998) “Establishment And Characterization Of A Mantle Cell
cytolysis only in the presence of cynolmolgus monkey s.
Lymphoma Cell Line,” Br. J. Haematol. 102(5):1323-1326) were incubated in the
presence of a DARTTM y and resting eral blood mononuclear cells
(PBMC) (E:T = 30:1). The results of this experiment (Figures 13A and 13B)
revealed that the CD19-h-mAb2 DARTTM y of the present invention cause
redirected T-cell-mediated killing that was equivalent to that observed using a CD19 x
CD3 DART diabody specific for human CD3. Thus the extension of specificity to
non-human CD3 holologs did not impair the y of the DARTTM diabody to
e redirected killing.
Example 14
Redirected Cytolysis by Cynomolgus Monkey Cross Reactive Dual Affinity Retargeting
Reagents (DARTTMs) ies Specific For CD3
The ability of the above-described CD19-h-mAb2 DARTTM diabody to cause
redirected -mediated killing in the presence of either human or cynolmolgus
monkey was investigated.
HT-29 human colon cancer cells (Marchis-Mouren, G. et al. (1988) “HT 29,
A Model Cell Line: Stimulation By The Vasoactive Intestinal Peptide (VIP); VIP
Receptor Structure And Metabolism,” Biochimie 70(5):663-671); Fogh, J. et al.
(1975) In: J. Fogh (ed.), HUMAN TUMOR CELLS IN VITRO, New York: Plenum Press.
1975) were incubated in the presence of human or cynolmolgus monkey T-cells (E:T
ratio = 30:1) and either the above-described CD19-h-mAb2 DARTTM diabody or a
CD19 x CD3 DART diabody whose CD3 sequences were derived from antibody FN-
18. Antibody FN—18 exhibits specificity only to cynolmolgus monkey CD3 (Nooij,
F.J. et al. (1986) “Difi’erentiation Antigens 0n Rhesus Monkey Lymphocytes. I.
Identification Of T Cells Bearing CD3 And CD8, And OfA Subset 0f CD8-Bearing
Cells,” Eur. J. Immunol. 16(8):975-979; Meng, G. et al. (1998) “The Efi’ect OfAnti-
munotoxin 0n T Lymphocyte Function in vitro,” Transpl. Immunol. 3-
59). The resultant percent cytotoxicity as a function of diabody concentration was
measured. The results (Figures 14A and 14B) show that the CD19-h-mAb2 DARTTM
diabody was able to mediate cytolysis in the ce of either human or non-human
T-cell effector cells. In contrast, the FN-18 diabody was capable of mediating
cytolysis only in the presence of cynolmolgus monkey T-cells.
U1 “11 Llwll Uxx _1_\_/\/11 L\UUV1}LU1 1J1 x1\1 uluuuu)’ \Uutjuulv UL Ulllullls LU LUL 1\
and the T-cell receptor). The ERBITUXTM-FNlS CD3 DARTTM y (capable of
Example 15
Dual Affinity eting Reagents (DARTTMs) Diabodies Require Target Cell
Engagement
In order to demonstrate that the observed redirected g mediated by the
CD3 DARTTM diabodies of the present invention was specific, the extent of g in
the presence and absence of target cells was determined.
Human PMBCs were ted in the presence of the above-described
ERBITUXTM-h-rnAbZ DARTTM y, an ERBITUXTM-T-Cell Receptor DARTTM
diabody (capable of binding to EGFR (Epidermal Growth Factor Receptor) and the T-
cell receptor), or an ERBITUXTM-FNlS CD3 DARTTM diabody (capable of binding
to EGFR and to cynolmolgus monkey CD3). The incubations were conducted in the
presence or absence of A498 kidney cancer target cells (Giard, DJ. et al. (1973) “in
vitro Cultivation Of Human Tumors.‘ Establishment 0f Cell Lines Derived From A
Series Of Solid Tumors,” J. Natl. Cancer Inst. 51:1417-1423; Fogh, J. (1978)
“Cultivation, Characterization, And Identification Of Human Tumor Cells With
Emphasis 0n Kidney, Testis And Bladder Tumors,” Natl. Cancer Inst. Monogr. 49:5-
The CD69 glycoprotein is an early activation antigen of T and B
lymphocytes that is expressed on cells of most hematopoietic lineages, ing
phils after stimulation (Atzenia, F. et al. (2002) “Induction 0fCD69 tion
Molecule 0n Human Neutrophils by GM-CSF, IFN-y, and IFN-a,” Cellular Immunol.
220(1): 20-29). The CD69 Mean Fluorescent Intensity (MFI) was therefore measured
(as a function of diabody concentration) as a means for assessing immune system
activation (see, e. g., Ampel, N.M. et al. (2002) “In Vitro Whole-Blood Analysis of
Cellular Immunity in Patients with Active Coccidioidomycosis by Using the Antigen
Preparation T2 7K,” Clinical Diagnostic Laboratory Immunology 9(5): 1039-1043).
] The results es 15A and 15B) show that immune system activation (as
measured by the MFI of CD69) increased only when CD4+ or CD8+ T cells were
incubated with the ERBITUXTM-h-mAbZ DARTTM diabody of the present ion
or an ERBITUXTM-T-Cell Receptor DARTTM diabody (capable of binding to EGFR
and the T-cell receptor). The ERBITUXTM-FNlS CD3 DARTTM diabody (capable of
adaptations 01 I116 111V611I1011 IOllOWlng, 111 general, I116 1311116113168 01 I116 111V611I1011 8.110,
including such departures from the present disclosure as come within known or
binding to EGFR and to cynolmolgus monkey CD3) failed to induce an increase in
the CD69 MFI.
Example 16
Redirected g by Humanized Cynomolgus Monkey / Human Cross-Reactive
DARTTM Diabodies
To further demonstrate the ability of the DARTTM diabodies of the present
invention to e redirected killing, A498 kidney cancer target cells or A431
moid carcinoma cells (Lee, C.M. et al. (2010) “The Distribution Of The
Therapeutic Monoclonal Antibodies Cetuximab And Trastuzumab Within Solid
Tumors,” BMC Cancer 10:25.5; pages l-l 1; Bryant, J.A. et al. (2004) “EGF tes
Intracellular And ellular Calcium Signaling By Distinct Pathways In Tumor
Cells,” Cancer Biol. Ther. 3(12):1243-1249) and the extent of redirected g mediated
by various DARTTM diabodies in the presence of PMBC effector cells (E:T = 30:1) was
determined.
Cells were incubated in the presence of either ERBITUXTM-h-mAb2
DARTTM diabody, ERBITUXTM-m-mAb2 DARTTM y or 4420-h-mAb2 DARTTM
diabody (negative control) or a l secondary dy. g to target cells was
determined by measuring MFI. Redirected killing was assessed by measuring the %
cytotoxicity.
The results of this investigation are shown in Figures 16A-16D. Diabodies
haVing specificity for CD3 and EGFR were found to be able to bind to A498 or A431
cells (Figure 16A and 16C, respectively), and to mediate redirected killing of these cells
e 16B and 16D, respectively).
All publications and patents mentioned in this specification are herein
incorporated by reference to the same extent as if each indiVidual publication or
patent application was specifically and individually indicated to be incorporated by
reference in its entirety. While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is capable of further
modifications and this application is intended to cover any variations, uses, or
adaptations of the invention ing, in general, the principles of the invention and
including such departures from the present disclosure as come within known or
customary practice Within the art to which the invention pertains and as may be
applied to the ial features hereinbefore set forth.
Claims (18)
- Claim 1. A CD3-binding molecule comprising an antigen-binding fragment of an antibody, wherein said antigen-binding fragment comprises an dy CD3-specific VL domain and an antibody CD3-specific VH domain, n said ecific VL domain and said CD3-specific VH domain form an antigen-binding domain capable of immunospecifically binding to both an epitope of human CD3 and to an epitope of the CD3 of a non-human mammal, wherein: (I) said CD3-specific VL domain is selected from the group consisting of h-mab2 VL-1 (SEQ ID NO:16), h-mab2 VL-2 (SEQ ID , h-mab2 VL-3 (SEQ ID NO:20), h-mab2 VL-4 (SEQ ID NO:22), h-mab2 VL-5 (SEQ ID NO:24), hmab2 VL-6 (SEQ ID NO:26), h-mab2 VL-7 (SEQ ID NO:28), h-mab2 VL-8 (SEQ ID NO:30), h-mab2 VL-9 (SEQ ID NO:32), and h-mab2 VL-10 (SEQ ID NO:34), and (II) said CD3-specific VH domain is selected from the group consisting of h-mab2 VH-1 (SEQ ID NO:36), h-mab2 VH-2 (SEQ ID NO:38), h-mab2 VH-3 (SEQ ID NO:40), h-mab2 VH-4 (SEQ ID NO:42), h-mab2 VH-5 (SEQ ID NO:44), hmab2 VH-6 (SEQ ID NO:46), h-mab2 VH-6L (SEQ ID NO:54), h-mab2 VH-7 (SEQ ID NO:48), h-mab2 VH-8 (SEQ ID NO:50), h-mab2 VH-8L (SEQ ID NO:55), h-mab2 VH-8 di-1 (SEQ ID NO:56), h-mab2 VH-8 di-2 (SEQ ID NO:57), hmab2 VH-6M (SEQ ID NO:72), h-mab2 VH-8M (SEQ ID NO:74), h-mab2 VH-2k (SEQ ID NO:87), and h-mab2 VH-5k (SEQ ID NO:88).
- Claim 2. The CD3-binding molecule of claim 1, wherein said CD3-specific VL domain is h-mab2 VL-6 (SEQ ID NO:26).
- Claim 3. The CD3-binding molecule of any of claims 1-2, n said CD3- specific VH domain is h-mab2 VH-8 (SEQ ID , h-mab2 VH-4 (SEQ ID NO:42), or h-mab2 VH-2k (SEQ ID NO:87).
- Claim 4. The nding molecule of any one of claims 1-3, wherein said molecule is an antibody.
- Claim 5 The CD3-binding antibody of claim 4, wherein said antibody lacks an Fc region or comprises an Fc region that: (A) lacks effector function or has d effector function; or (B) impairs the y of the Fc region of said antibody to bind to an Fc receptor; 2176189v1 wherein said reduction in effector function and said impairment of binding ability is relative to that of a wild-type Fc receptor.
- Claim 6. The CD3-binding molecule of any of claims 1-3, n said le is a CD3-binding y that comprises a first polypeptide chain and a second polypeptide chain, said chains being covalently bonded to one r, wherein: (I) said first polypeptide chain comprises an amino terminus and a carboxy terminus and from N-terminus to C-terminus: (i) a domain (A) sing said CD3-specific VL domain; (ii) a domain (B) comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2); and (iii) a domain (C); wherein said s (A) and (B) do not associate with one another to form an epitope binding site; (II) said second polypeptide chain comprises an amino terminus and a carboxy terminus and from N-terminus to inus: (i) a domain (D) comprising a binding region of a light chain variable domain of said second immunoglobulin (VL2); (ii) a domain (E) comprising said CD3-specific VH domain; (iii) a domain (F); wherein said domains (D) and (E) do not associate with one another to form an epitope binding site; and wherein: (1) said domains (A) and (E) associate to form said antigen-binding domain that is capable of immunospecifically binding to both human CD3 and to the CD3 of a non-human mammal; (2) said domains (B) and (D) associate to form a binding site that immunospecifically binds to a second epitope, said second epitope being different from the CD3 epitope bound by the antigen-binding domain formed from said association of said domains (A) and (E); and (3) said domains (C) and (F) are covalently associated together.
- Claim 7. The CD3-binding diabody of claim 6, wherein said second e is not an e of CD3. 2176189v1
- Claim 8. The CD3-binding diabody of claim 6, wherein said second epitope is an epitope of CD3 that is different from the CD3 epitope bound by the antigen-binding domain formed from said association of said domains (A) and (E).
- Claim 9. The CD3-binding molecule of any one of claims 1-3, the antibody of any of claims 4-5, or the diabody of any of claims 6-8 which is humanized.
- Claim 10. The CD3-binding molecule of any one of claims 1-3 or 9 or the diabody of any one of claims 6-9 which is capable of immunospecifically binding to CD3 and to scein.
- Claim 11. The CD3-binding le of any one of claims 1-3 or 9 or the y of any of claims 6-9, which is e of immunospecifically binding to both: (i) CD3 and (ii)(a) a tumor antigen, or (ii)(b) a cell surface antigen, receptor or receptor ligand.
- Claim 12. The nding molecule or diabody of claim 11, wherein said molecule or diabody is capable of immunospecifically binding to CD3 and to a tumor antigen sed on a tumor cell, wherein said tumor cell is a tumor cell from a cancer selected from the group consisting of: breast cancer, prostate cancer, gastric cancer, lung cancer, stomach cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, ovarian cancer, oral cavity cancer, pharyngeal cancer, esophageal cancer, eal cancer, bone cancer, skin cancer, melanoma, uterine cancer, testicular cancer, r cancer, kidney cancer, brain cancer, astoma, thyroid cancer, lymphoma, myeloma, and leukemia.
- Claim 13. The CD3-binding molecule or diabody of claim 11, wherein said molecule or diabody is capable of immunospecifically binding to CD3 and to a cell surface antigen, or or receptor ligand, wherein said cell surface antigen, receptor or receptor ligand is HER2/neu, B7-H3, CD20, PSMA, IGF-1R., Ep-CAM, or is a molecule ed in a T cell – B cell association that leads to T cell or B cell activation in an ve immune response.
- Claim 14. The CD3-binding molecule or diabody of claim 13, wherein said molecule or diabody is capable of immunospecifically binding to CD3 and to a molecule involved in said T cell – B cell association and said molecule involved in said T cell – B cell association is selected from the group consisting of CD19, CD20, CD22, CD23, CD27, CD32B, CD38, CD40, CD79a, CD79b, CD80, CD86, LFA-I, LFA-3 and CFAI. 2176189v1
- Claim 15. A pharmaceutical composition comprising the CD3-binding molecule of any of claims 1-3 or 9-14, the antibody of any one of claims 4-5 , or the diabody of any one of claims 6-14 , and a pharmaceutically acceptable carrier, excipient or diluent.
- Claim 16. The pharmaceutical composition of claim 15, for use in the treatment of cancer or an autoimmune or matory e.
- Claim 17. The pharmaceutical composition of claim 16, for use in the treatment of an autoimmune or inflammatory disease selected from the group consisting of: type I n-dependent diabetes, rheumatoid tis, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease, myasthenia gravis, celiac’s disease, Sjogren's syndrome, Grave’s disease, Crohn’s disease, autoimmune hepatitis, psoriasis, tic arthritis, asthma, ic rhinitis, effects from organ transplantation, or graft vs. host disease (GVHD).
- Claim 18. The pharmaceutical composition of claim 16, for use in the treatment of type I insulin-dependent diabetes.
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NZ703939A NZ703939A (en) | 2011-05-21 | 2012-05-16 | Cd3-binding molecules capable of binding to human and non-human cd3 |
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US201161488716P | 2011-05-21 | 2011-05-21 | |
US61/488,716 | 2011-05-21 | ||
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US61/530,353 | 2011-09-01 | ||
PCT/US2012/038219 WO2012162067A2 (en) | 2011-05-21 | 2012-05-16 | Cd3-binding molecules capable of binding to human and non-human cd3 |
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