EA045234B1 - ANTIBODIES BINDING TO A COMPLEX INCLUDING TRIMERIC TNFα, THEIR APPLICATION AND ANTIBODY-ENCODING POLYNUCLEOTIDE - Google Patents

Description

Field of the present invention

The present invention provides antibodies that can be used to screen for small molecule modulators of the TNF superfamily and that form complexes with members of the TNF superfamily. First of all, the invention relates to antibodies that selectively bind to such complexes, and to the use of such antibodies. The present invention also provides assay methods for identifying novel modulators of the TNF superfamily using such antibodies.

Background of the present invention

The tumor necrosis factor (TNF) superfamily is a family of proteins that exhibit general regulatory function for cell survival and cell death. Members of the TNF superfamily are characterized by a common amino acid motif at the core of the molecule, which consists of two antiparallel β-sheet sheets with antiparallel β-strands forming a roll-type β-structure. Another common feature of members of the TNF superfamily is the formation of homo- or heterotrimeric complexes. It is these trimeric forms of TNF superfamily members that bind to and activate specific TNF superfamily receptors.

TNFa is the original member of the TNF superfamily. Dysregulation of TNFa production is involved in the development of a number of pathological conditions of great importance in medicine. For example, TNFa is involved in the development of rheumatoid arthritis, inflammatory bowel disease (including Crohn's disease), psoriasis, Alzheimer's disease (AD), Parkinson's disease (PD), pain, epilepsy, osteoporosis, asthma, systemic lupus erythematosus (SLE) and multiple sclerosis (MS). Other members of the TNF superfamily are also involved in the development of pathological conditions, including autoimmune diseases.

Standard antagonists of members of the TNF superfamily are high molecular weight and act by inhibiting the binding of a member of the TNF superfamily to its receptor. Examples of standard antagonists include anti-TNFa antibodies, primarily monoclonal antibodies such as infliximab (Remicade®), adalimumab (Humira®) and certolizumab pegol (Cimzia®), or soluble TNFa receptor fusion proteins such as etanercept (Enbrel® ).

Brief Description of the Present Invention

The present inventors have identified classes of small molecule compounds (SMEs) that modulate TNFa. These compounds act upon binding to the homotrimeric form of TNFa and lead to the induction and/or stabilization of a conformational change in the homotrimer structure. For example, bound TNFa homotrimers can bind to TNFa receptors but are less or not able to induce signaling downstream TNFa receptor signaling pathways. These compounds can be used in the treatment of TNFa-mediated conditions.

The present inventors have also constructed antibodies that selectively bind to complexes including such compounds and members of the TNF superfamily. These antibodies can be used to identify other compounds that are capable of inhibiting TNFa in this manner, and can also be used as biomarkers of target binding.

Accordingly, the present invention provides an antibody that selectively binds to a complex comprising (i) trimeric TNFa, and (ii) a compound selected from the group consisting of compounds (1)-(5) or salts or solvates thereof:

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where the antibody includes a combination of sequences

HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 SEQ ID NOs: 4/5/6/1/2/3.

Said antibody preferably contains a pair of sequences HCVR and LCVR SEQ ID NO: 8 and 7.

In another preferred embodiment, it contains a heavy chain of SEQ ID NO: 12 or 13 and a light chain of SEQ ID NO: 11.

Another embodiment of the present invention is an antibody that selectively binds to a complex containing (i) trimeric TNFa, and (ii) a compound selected from the group consisting of compounds (1)-(2) or salts, or solvates thereof:

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or

where the antibody contains a combination of sequences

HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 SEQ ID NO: 19/20/21/1/17/18.

Preferably, said antibody contains a pair of sequences HCVR and LCVR SEQ ID NO: 23 and 22.

Preferably it contains a heavy chain of SEQ ID NO: 27 or 28 and a light chain of SEQ ID NO: 26.

The subject antibodies preferably bind selectively to the trimer-compound complex as compared to binding to the compound in the absence of the TNFa trimer and/or to the compound in the absence of the TNFa trimer.

Moreover, the K _D-ab value when binding an antibody to the trimer-compound complex can decrease by at least 100 times, or at least 200 times compared to the K _D-ab value for binding to the TNFa trimer in the absence of a compound and/or for binding to the compound in the absence of TNFa.

Antibodies according to the invention may be:

(a) a humanized antibody; or (b) a Fab fragment, modified Fab, Fab', modified Fab', F(ab') ₂ , Fv, single domain antibody, or scFv.

Another object of the present invention is a polynucleotide encoding the above antibodies.

Another aspect of the present invention is the use of the claimed antibodies as a biomarker of target binding to detect a compound-trimer complex in a sample obtained from a subject, wherein said antibody is detectable, and wherein said complex includes trimeric TNFa and a compound that is capable of binding to trimeric TNFa, and thereby the compound-trimer complex binds to the desired receptor with TNF and modulates trimer-induced signal transduction through the receptor. In addition, the present invention provides a method for detecting the interaction of a target with a TNFa trimer compound, whereby the trimer compound complex binds to the cTNF receptor and modulates trimer-induced signal transduction through the receptor, the method comprising:

(a) obtaining a sample from a subject who has been administered said compound, (b) contacting said antibody with said sample and a control sample, wherein said antibody is detectable, (c) determining the level of binding of said detectable antibody to said sample and said control sample, wherein the binding of said detectable antibody to said sample is higher than the binding of said detectable antibody to said control sample, indicating interactions of the target with said compound upon binding to said TNFa trimer.

Another aspect described in this invention is the use of the above antibodies to detect a compound that causes a conformational change in the TNFa trimer, wherein said conformational change modulates signaling through the TNF receptor upon binding to the TNFa trimer.

Finally, the last aspect of the present invention is a method for identifying a compound capable of binding to TNFa and modulating TNFa trimer signaling through the receptor, which includes the following steps:

(a) performing a binding assay to measure the binding activity of the compound-trimer complex of interest TNFa and the compound of interest to the antibody, (b) comparing the binding activity measured in step (a) with the binding activity of various compound-trimer complexes for which it is known that they bind with high activity to the antibody specified in step (a), and (c) selecting the compound present in the compound-trimer complex in step (a) if the measured binding activity is acceptable from the comparison data specified in step ( b),

In a preferred embodiment, the method is a high speed analysis method.

Brief description of the figures

In fig. Figure 1 shows the crystal structure of human TNFa, in which residues N168, 1194, F220 and A221 are highlighted.

In fig. Figure 2 shows the results of HPLC chromatography of the CA185_01974 mFab fragment and compound (1). The peaks correspond to excess Fabs of 1.5x and 2.0x. Thus, the stoichiometric composition of 1 Fab: 1 TNFa was determined for the trimer.

In fig. Figure 3 shows the results of HPLC chromatography of the CA185_01979 mFab fragment and compound (1). Again, the peaks correspond to excess Fabs of 1.5x and 2.0x. Thus, the stoichiometric composition of 1 Fab: 1 TNFa was determined for the trimer.

In fig. Figure 4 presents the results of a summary analysis of TNFa-ELISA using compounds (3), (4) and (5) and commercial polyclonal anti-TNFa antibodies.

In fig. Figure 5 shows the results of a conformation-specific TNFa-ELISA analysis using the CA185_01974.0 antibody and compounds (3), (4) and (5). In this assay, no signal is observed for apoTNFa, indicating the specific nature of the binding of antibody CA185_01974 to the TNFa-related compound.

In fig. Figure 6 shows graphs of FACS histograms after staining with antibodies CA185_01974 and CA185_01979 at a concentration of 1 and 10 μg/ml. These graphs indicate that the antibodies only recognize TNFa that has been preincubated with compound (1). In the case of the DMSO control, no coloring is observed.

In fig. 7 shows FACS histogram plots after staining with CA185_01974 antibodies for the parental NS0 cell line and the engineered NS0 cell line overexpressing membrane TNFa. Cells were incubated with compound (1) or DMSO and developed with Fab antibody fragment. Again, the results show no staining with the DMSO control (both parental and engineered cell lines). However, in the presence of compound (1), coloration is observed in the engineered cell line.

In fig. 8 shows sensorgrams for determining affinity values for the CA185_01974 antibody using cynomolgus TNFa. Cynomolgus TNFa and DMSO were used as controls (top graphs). The lower graphs show replicate experiments for the cynomolgus TNFa complex with compound (4).

In fig. 9 shows sensorgrams for determining affinity values for the antibody CA185_01974 using human TNFa. Human TNFa and DMSO were used as controls (top graphs). The lower graphs show replicate experiments for human TNFa complex with compound (4).

In fig. 10 shows the structures of compounds (1)-(6).

Brief description of the sequence listing

SEQ ID NO: 1 represents the LCDR1 sequence of antibody CA185_01974.0.

SEQ ID NO: 2 represents the LCDR2 sequence of antibody CA185_01974.0.

SEQ ID NO: 3 represents the LCDR3 sequence of antibody CA185_01974.0.

SEQ ID NO: 4 represents the HCDR1 sequence of antibody CA185_01974.0.

SEQ ID NO: 5 represents the HCDR2 sequence of antibody CA185_01974.0.

SEQ ID NO: 6 represents the HCDR3 sequence of antibody CA185_01974.0.

SEQ ID NO: 7 represents the amino acid sequence of the LCVR antibody CA185_01974.0.

SEQ ID NO: 8 represents the amino acid sequence of the HCVR antibody CA185_01974.0.

SEQ ID NO: 9 represents the nucleotide (DNA) sequence of the LCVR antibody CA185_01974.0.

SEQ ID NO: 10 represents the nucleotide (DNA) sequence of the HCVR antibody CA185_01974.0

SEQ ID NO: 11 represents the amino acid sequence of the kappa light chain of the antibody

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CA185_01974.0.

SEQ ID NO: 12 represents the amino acid sequence of the antibody heavy chain mIgGI

CA185_01974.0.

SEQ ID NO: 13 represents the amino acid sequence of mFab (without the hinge region) of the heavy chain of the antibody CA185_01974.0.

SEQ ID NO: 14 represents the nucleotide (DNA) sequence of the kappa light chain of the antibody CA185_01974.0.

SEQ ID NO: 15 represents the nucleotide (DNA) sequence of mIgG1 heavy chain antibody CA185_01974.0.

SEQ ID NO: 16 represents the nucleotide (DNA) sequence of the mFab (without the hinge region) heavy chain of the antibody CA185_01974.0.

SEQ ID NO: 17 represents the LCDR2 sequence of antibody CA185_01979.0.

SEQ ID NO: 18 represents the LCDR3 sequence of antibody CA185_01979.0.

SEQ ID NO: 19 represents the HCDR1 sequence of antibody CA185_01979.0.

SEQ ID NO: 20 represents the HCDR2 sequence of antibody CA185_01979.0.

SEQ ID NO: 21 represents the HCDR3 sequence of antibody CA185_01979.0.

SEQ ID NO: 22 represents the amino acid sequence of the LCVR antibody CA185_01979.0.

SEQ ID NO: 23 represents the amino acid sequence of the HCVR antibody CA185_01979.0.

SEQ ID NO: 24 represents the nucleotide (DNA) sequence of the LCVR antibody CA185_01979.0.

SEQ ID NO: 25 represents the nucleotide (DNA) sequence of the HCVR antibody CA185_01979.0.

SEQ ID NO: 26 represents the amino acid sequence of the kappa light chain of antibody CA185_01979.0.

SEQ ID NO: 27 represents the amino acid sequence of mIgG1 heavy chain antibody CA185_01979.0.

SEQ ID NO: 28 represents the amino acid sequence of mFab (without the hinge region) of the heavy chain of the antibody CA185_01979.0.

SEQ ID NO: 29 represents the nucleotide (DNA) sequence of the kappa light chain of the antibody CA185_01979.0.

SEQ ID NO: 30 represents the nucleotide (DNA) sequence of mIgG1 heavy chain CA185_01979.0.

SEQ ID NO: 31 represents the nucleotide (DNA) sequence of the mFab (without the hinge region) heavy chain of the antibody CA185_01979.0.

SEQ ID NO: 32 represents the amino acid sequence of rat TNFa.

SEQ ID NO: 33 represents the amino acid sequence of mouse TNFa.

SEQ ID NO: 34 represents the amino acid sequence of human TNFa.

SEQ ID NO: 35 represents the amino acid sequence of the soluble form of human TNFa.

SEQ ID NO: 36 represents the amino acid sequence of the soluble form of human TNFa, but without the original S residue (which is a cloning error of the sequence SEQ ID NO: 35).

Detailed Description of the Present Invention

Modulators of members of the TNF superfamily.

The present inventors have identified test compounds that bind to the trimeric forms of members of the TNF superfamily. These compounds may be small molecular weight compounds (SME) with a molecular weight of 1000 Da or less, generally 750 Da or less, more preferably 600 Da or less. Their molecular weight may range from about 50 to about 1000 Da, or from about 100 to about 1000 Da. These compounds stabilize the conformation of a trimeric member of the TNF superfamily, which binds to an essential TNF superfamily receptor and modulates signal transduction through the receptor. Examples of such compounds include compounds of formulas (1)-(6).

The stabilizing effect of these compounds on the trimeric forms of members of the TNF superfamily can be quantified by measuring the midpoint temperature transition ( _Tm ) of the trimers in the presence and absence of the compound. T _m means the temperature at which 50% of biomolecules are characterized by an unfolded structure. Compounds that stabilize the trimers of members of the TNF superfamily increase the T _m of the trimers. T _m can be determined using an appropriate method known in the art, for example, using differential scanning calorimetry (DSC) or thermal denaturation method with a fluorescent probe.

The compounds may bind in the inner core space of the TNF superfamily member trimer (ie, the core of the trimer).

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These compounds can convert a member of the TNF superfamily into a TNF superfamily receptor antagonist. Therefore, these compounds are able to block signaling through a member of the TNF superfamily without competing with the high affinity interaction between the member of the TNF superfamily and its receptor.

They can stabilize the conformation of a trimeric member of the TNF superfamily, which binds to an essential TNF superfamily receptor and enhances signal transduction through the receptor. Therefore, these compounds are able to enhance signaling through a member of the TNF superfamily without competing with the high affinity interaction between the member of the TNF superfamily and its receptor.

Where compounds are described herein as antagonists, it is understood that the compounds may equally be agonists and enhance signaling involving the TNF superfamily receptor that binds to the trimeric TNF superfamily member complex, and thus may be agonist compound. Likewise, if antagonistic compounds are indicated in another context, methods for identifying such compounds and the use of such compounds may be used herein to apply equally to agonist compounds.

The compounds described in this context are allosteric modulators that bind to natural agonists of TNF superfamily receptors, that is, trimeric forms of members of the TNF superfamily, and which cause these trimers to adopt a conformation that still retains the ability to bind to the desired receptor TNF superfamily and modulate signal transduction through the receptor. It should be understood that the term modulation means that the compound may have an antagonistic effect, that is, reduce signaling through a TNF superfamily receptor, or have a stimulatory effect, that is, increase or enhance signaling through a TNF superfamily receptor.

These compounds can convert natural agonists of members of the TNF superfamily into antagonists. Conversely, standard antagonists of TNF superfamily members bind to a TNF superfamily member or a TNF superfamily receptor and prevent the TNF superfamily member from binding to the desired receptor. They may increase signaling through a TNF superfamily receptor when a TNF superfamily member is in a bound state compared to the level of signaling through a TNF superfamily receptor when a TNF superfamily member is in a bound state in the unbound state. Therefore, the compounds can convert natural agonists of members of the TNF superfamily into so-called superagonists. Therefore, the compounds may be known as allosteric modulators of ligand activity (AMLA).

The compounds are not limited as to their chemical formula or structure, provided that they bind to at least one member of the TNF superfamily and stabilize the conformation of a trimeric member of the TNF superfamily that binds to the desired TNF superfamily receptor and modulates signal transduction through the TNF superfamily receptor. Therefore, compounds can be identified using antibodies and methods described in this context. The compounds may include a benzimidazole moiety or isosteric analogues thereof.

The compounds can increase the binding affinity of TNF superfamily members (in compound-trimer complex form) for the desired receptor compared to the binding affinity of TNF superfamily members for the desired receptor in the absence of the compounds.

The compounds bind to the trimeric forms of members of the TNF superfamily. Such compounds bind specifically (or selectively) to the trimeric forms of one or more members of the TNF superfamily. A compound may bind specifically (selectively) to only one member of the TNF superfamily, but not to any other member of the TNF superfamily. The compound may also bind specifically to two, three, four, or up to all members of the TNF superfamily. It should be understood that the term specifically (or selectively) means that the compounds bind to the molecule or molecules of interest, in which case the trimeric form of a member of the TNF superfamily is characterized by the absence of significant cross-reactivity with any other molecule, which may include other members of the TNF superfamily. Cross-reactivity can be assessed by any suitable method, for example surface plasmon resonance. Cross-reactivity of a compound against the trimeric form of a member of the TNF superfamily with a molecule that differs from the trimeric form of that particular TNF superfamily member can be considered significant if the binding of the compound to another molecule is at least 5, 10, 15, 20, 25 higher. 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 100% compared to the binding of the compound to the trimeric form of the TNF superfamily member of interest. For example, cross-reactivity may be considered significant if the degree of binding of a compound to another molecule increases by an amount ranging from about 5% to about 100%, typically from about 20% to about 100%, or about 50% to about 100%, according to compared to the binding of the compound to the trimeric form of the TNF superfamily member of interest. A compound that is specific (or selective) for the trimeric form of a member of the super-TNF family can bind to another molecule, with the degree of binding reduced by approximately 90, 85, 80, 75, 70, 65, 60, 55 , 50, 45, 40, 35, 30, 25 or 20% compared to the binding of the compound to the trimeric form of the TNF superfamily member of interest (up to zero binding). If the compound is suitably bound to another molecule, the degree of binding is reduced by about 20%, about 15%, about 10%, or about 5%, about 2%, or about 1% compared to binding of the trimeric form of the compound of the TNF superfamily member under study (before zero binding).

The rate at which a test compound binds to a member of the TNF superfamily is in this context called the rate of association or on-rate, or k _on - _c , and the rate at which the test compound dissociates, that is, separates from a member of the TNF superfamily, is called the dissociation rate or off-rate or k _off . _{c_} . As used herein, the symbol K _Dc denotes the binding affinity (dissociation constant) of the test compound for a member of the TNF superfamily. K _D _ _c means k _off . _c /k _on . _c . The test compounds can reduce the on-rate, which can be measured in minutes by mass spectrometric analysis based on the peak intensity of the compound-trimer complex of a member of the TNF superfamily. K _{D_c} values for a test compound can be estimated by repeating this measurement in the presence of different ratios of TNF superfamily member/compound- _trimer complex. Typically, the binding of the compounds described in the invention to trimers of the TNF superfamily is characterized by high rates of association, ideally at the level of 10 ⁷ M ^-1 s ^-1 , and low rates of dissociation, for example, at the level of 10 ^-3 s ^-1 , 10 ^-4 s ^-1 , or the dissociation rate is not measurable.

As used herein, the symbol ko _n _ _r denotes the rate of association with which the compound-trimer complex binds to the TNF superfamily receptor. As used herein, the symbol ko _ff _ _r denotes the dissociation rate at which the compound-trimer complex dissociates, that is, separates from the TNF superfamily receptor. As used herein, the symbol KD-r denotes the binding affinity (dissociation constant) of the compound-trimer complex for the receptor superfamily. The symbol K _Dr means k _off-r /k _on-r .

The K _Dr value for a member of the TNF superfamily when bound to its receptor in the presence of the test compound (i.e., in the form of a compound-trimer complex) can be reduced by at least approximately 1.5-fold, 2-fold, 3-fold, 4-fold, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, compared to the K _D value _ _r for a member of the TNF superfamily upon binding to its receptor in the absence of the test compound. The K _Dr value of a compound-trimer complex when bound to a member of the TNF superfamily can be reduced by at least about 1.5-fold, generally at least about 3-fold, more preferably at least about 4-fold, compared to the KD value -r to bind a TNF superfamily trimer to a TNF superfamily receptor in the absence of a test compound, that is, the binding affinity of the TNF superfamily compound-trimer complex can be increased by at least about 1.5-fold, generally by at least about 3-fold, more preferably at least about 4-fold compared to the binding affinity of the TNF superfamily trimer in the absence of the test compound.

A compound described herein can increase the binding affinity of a member of the TNF superfamily for its receptor by approximately 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold , 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more compared to the binding affinity of a TNF superfamily member for its receptor in the absence of the test compound.

Binding affinity can be expressed as a binding affinity constant (KD-r), as well as in any other units such as µM, nM or pM. The lower the K _D - _r value, the greater the binding affinity of a member of the TNF superfamily for its receptor.

The K _D _ _r value for a member of the TNF superfamily when binding to its receptor in the presence of the test compound can be reduced by approximately 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or more the K _Dr value for binding of a TNF superfamily member to its receptor in the absence of the test compound.

A decrease in the K _Dr value for a compound-trimer complex upon binding to a TNF superfamily receptor compared to the K _Dr value for only one TNF superfamily trimer with a TNF superfamily receptor may occur as a result of an increase in the rate of binding kon.r of the compound-trimer complex to a TNF superfamily receptor by compared to only one trimer of the TNF superfamily, or as a result of a decrease in the dissociation rate compared to only one trimer of the TNF superfamily. In general, the rate of kon.r binding of the compound-trimer complex to the TNF superfamily receptor is increased compared to the trimer alone. In general, the rate of koff-r binding of the compound-trimer complex to the TNF superfamily receptor is reduced compared to trimer alone. The most preferential kon-r binding of the compound-trimer complex to the TNF superfamily receptor increases, and the rate of k _off - _r binding of the complex to

- 7 045234 the union-trimer with the TNF superfamily receptor is reduced compared to the TNF superfamily trimer alone. The k _on-r value for binding the compound-trimer complex to the desired TNF superfamily receptor can be increased by at least about 1.5-fold, or at least about 2-fold, and preferably at least about 3-fold, compared to the ko value _n . _r for binding of the trimer to its receptor in the absence of the compound and/or the k _off - _r value for binding of the compound-trimer complex to the desired TNF superfamily receptor can be reduced by at least about 1.2-fold, at least about 1.6-fold , at least about 2-fold, more preferably at least about 2.4-fold, compared to the koff-r value for binding of a TNF superfamily trimer to its receptor in the absence of a compound.

The rate of on-rate binding of a compound to a TNF superfamily trimer (k _on - _c ) is generally higher than the rate of on-rate binding of a compound-trimer complex to a TNF superfamily receptor (ko _n _ _r ). The off-rate for binding a compound-trimer complex to a TNF superfamily receptor (k _of f- _r ) is also generally higher than the off-rate for binding a compound to a TNF superfamily trimer (k _off - _c ). Most preferably, the on-rate rate of binding of a compound to a TNF superfamily trimer (ko _n . _c ) is generally greater than the on-rate rate of binding of a compound-trimer complex to a TNF superfamily receptor (ko _n „ _r ), and the off-rate rate for binding The complex of the compound trimer with the TNF superfamily receptor (k _of f_ _r ) is higher than the off-rate for binding the compound with the TNF superfamily trimer (koff- _c ). The _{KD_c} value for the binding _of a compound to a TNF superfamily trimer is generally lower than the _{KD_r} value for the binding of a compound-trimer complex to _a TNF superfamily receptor, that is, the compound has higher binding activity for the trimer than the compound-trimer complex in relation to the receptor.

The values of ko _n _ _r , k _off _ _r and K _D _ _r for both the compound-trimer complex and the TNF superfamily trimer in relation to the desired TNF superfamily receptor can be determined using an appropriate method, e.g. surface plasmon resonance, mass spectrometry and isothermal calorimetry. The K _{D_r} value of a TNF superfamily member for binding to its receptor in the presence of a test compound may be 1 μM, 100 nM, 10 nM, 5 nM, 1 nM, 100 pM, 10 pM or less (usually down to a lowest value _of approximately 1 pM). The K _{D_r} value of a TNF superfamily member for binding to its receptor in the presence of a test compound ( _ie , as part of a compound-trimer complex) may be 1 nM or less. The K _{D_r} value of the compound-trimer complex for binding to the desired TNF superfamily receptor may be less than 600 pM, more preferably less than 500 pM, less than 400 pM, less than 300 pM, less than 200 pM _, less than 100 pM, or less than 50 pM (again up to to the lowest value 1 pM). The K _{D_r} value of the compound-trimer complex for binding to the desired TNF superfamily receptor may _be less than about 200 pM (up to about 1 pM).

Compounds can be identified by an assay that involves determining the _{KD_r} value of the trimeric form of a TNF superfamily member in a sample _of the TNF superfamily member and the compound by comparing the _{KD_r} value _of the trimeric form of a TNF superfamily member in the sample with a control sample, and selecting the compound.

The compounds stabilize the trimeric form of a member of the TNF superfamily. Stabilization is believed to occur if the test compound increases the proportion of trimer compared to the amount of trimer observed in a sample containing a member of the TNF superfamily and a destabilizing agent in the absence of the test compound. The test compound can increase the amount of trimer by approximately 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400% or more compared to the amount of trimer present in a sample containing a member of the TNF superfamily and a destabilizing agent in the absence of the test compound.

The test compound may also increase the amount of trimer compared to the amount observed in a sample of the TNF superfamily member in the absence of both the destabilizing agent and the test compound. The test compound can increase the amount of trimer by approximately 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400% or more compared to the amount of trimer present in a sample containing a member of the TNF superfamily in the absence of both components, the destabilizing agent and the test compound.

The test compound can increase the amount of TNF superfamily member bound to its receptor by approximately 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90. 95, 100, 150, 200, 300, 400% or more compared to the amount of the TNF superfamily member bound to its receptor in a sample containing the TNF superfamily member in the absence of the test compound.

The test compound may increase the stability of the trimeric form of a member of the TNF superfamily. An increase in the stability of the trimeric form of a TNF superfamily member is believed to occur if the compound of interest increases the average thermal transition temperature ( _Tm ) of the trimeric form of the TNF superfamily member compared to the _{Tm of} the trimeric form of the TNF superfamily member observed for a sample containing the TNF superfamily member and the destabilizing agent is absent

- 8 045234 vii of the test compound. The Tm of the trimeric form of a member of the TNF superfamily is the temperature at which 50% of the biomolecules are in the unfolded state. The Tm of the trimeric form of a member of the TNF superfamily in the presence and/or absence of a test compound can be measured using any appropriate method known in the art, for example, using differential scanning calorimetry (DSC) or thermal denaturation with a fluorescent probe.

The test compound can increase the Tm value of the trimeric form of a member of the TNF superfamily by at least 1°C, at least 2°C, at least 5°C, at least 10°C, at least 15°C , at least 20°C or more compared to the Tm value of the trimeric form of the TNF superfamily member in a sample containing the TNF superfamily member in the absence of the test compound. The test compound can increase the Tm value of the trimeric form of a member of the TNF superfamily by at least 1°C, typically by at least 10°C, and more preferably in the range of 10°C to 20°C.

The compounds can completely or partially inhibit signaling through the TNF receptor if a member of the TNF superfamily, in the form of a compound-trimer complex, binds to the receptor. The compound may be effective by reducing TNF superfamily receptor signaling by at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%. In another embodiment, compounds can enhance signaling through the TNF receptor if a member of the TNF superfamily, in the form of a compound-trimer complex, binds to the receptor. The compound may also be effective by increasing TNF superfamily receptor signaling by at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 200%. Any change in the level of signal transduction can be measured by any appropriate method, including measurement of reporter gene activity using alkaline phosphatase or luciferase, NF-κB translocation using systems such as Cellomics Arrayscan, phosphorylation of effectors located in downstream signaling pathways, influx of compounds associated with signal transmission, or cell death.

The compounds can modulate at least one of the signal transduction effects observed in the following signal transduction pathways through the TNF receptor if a member of the TNF superfamily, in the form of a compound-trimer complex, binds to the receptor. Such effects are discussed in this context and include TNF superfamily-induced production of IL-8, IL17A/F, IL2, and VCAM, TNF superfamily-induced NF-κB activation, and neutrophil influx. Standard methods are known in the art for measuring the effects of TNF superfamily members observed in the following signaling pathways. The compounds may modulate at least 1, 2, 3, 4, 5, 10, or up to all of the TNF receptor signaling effects observed in the following signaling pathways.

The activity of compounds can be quantified using standard terminology such as IC ₅₀ or half maximum effective concentration (EC ₅₀ ) values. IC ₅₀ values indicate the concentration of a compound that is required to produce 50% inhibition of a specific biological or biochemical function. IC ₅₀ values indicate the concentration of a compound that is required to achieve 50% of its maximum effect. The IC ₅₀ or EC ₅₀ values of compounds can be 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM , 5 nM, 1 nM, 100 pM or less (down to a lowest value of approximately 10 pM or 1 pM).

The IC ₅₀ and EC ₅₀ values can be measured using any appropriate method, for example, cytokine production can be quantified by ELISA. The IC ₅₀ and EC ₅₀ values can be estimated using a standard 4-parameter logistic model known as the sigmoid dose-response model.

As stated above, examples of compounds that are capable of binding to TNF and modulating signal transduction include compounds of formulas (1)-(6).

TNF superfamily member modulator complexes.

The present inventors have found that binding of the compounds described herein to the trimeric forms of members of the TNF superfamily results in a change in the conformation of the TNF superfamily trimer. First, the conformation of the trimer of a member of the TNF superfamily is deformed or distorted upon binding to a compound as described above.

For example, if compounds (1)-(6) bind to the soluble domain of TNFa, TNF retains its trimeric structure, but the A and C subunits move away from each other and the C subunit rotates to form a pocket between these subunits.

Without being limited by theory, it is believed that in the absence of coupling, trimeric members of the TNF superfamily, including TNFa trimers, are capable of binding to three separate dimers of TNF superfamily member receptors. Each TNF superfamily member receptor dimer is capable of binding to two separate TNF superfamily trimers. This binding results in the aggregation of multiple TNF superfamily trimeric members and TNF superfamily member receptor dimers, creating a signaling block that initiates signal transduction in downstream signaling pathways.

If trimeric TNFa binds to a compound, the conformation of the resulting complex undergoes deformation. Accordingly, without being limited by theory, it is believed that in the presence of a compound described herein, trimeric members of the TNF superfamily, including trimers of TNFa, are capable of binding to only two distinct receptor dimers of a member of the TNF superfamily. The fact that only two, rather than three, individual TNF superfamily member receptor dimers bind to a trimeric TNF superfamily member results in reduced or inhibited aggregation of multiple trimeric TNF superfamily members and TNF superfamily member receptor dimers. This phenomenon reduces or inhibits the formation of signaling blocks and thus reduces or inhibits signal transduction in downstream signaling pathways.

The antibodies described herein can be used to detect members of the TNF superfamily with a deformed conformation that results from binding of a compound as described herein. Typically, a member of the TNF superfamily with a deformed or disrupted conformation is a trimeric member of the TNF superfamily. However, these antibodies can also bind to other forms of the TNF superfamily member. For example, they can bind to monomers of the TNF superfamily.

Typically, a member of the TNF superfamily is TNFa and may be trimeric TNFa (primarily TNFa _s ).

Accordingly, the invention provides a complex comprising a trimeric protein that is a member of the TNF superfamily and a compound associated therewith, whereby the trimeric complex binds to a desired TNF superfamily receptor and modulates signal transduction induced by the trimer through the receptor, wherein said complex binds to an antibody with an affinity of at least 1 nM (ie, 1 nM or less, down to about 1 pM). Typically, a member of the TNF superfamily is TNFa, most notably TNFa _s .

Moreover, the antibody generally binds to the complex with an affinity that is reduced by at least about 100-fold (affinity is improved by at least about 100-fold), preferably by about 200-fold, compared to the binding affinity to the compound in the absence of the TNF trimer and/or binding to the TNF trimer in the absence of a compound.

The invention also describes a compound that is capable of binding to a trimeric protein that is a member of the TNF superfamily, thereby forming a complex, and thereby the compound-trimer complex binds to the desired TNF superfamily receptor and modulates trimer-induced signal transduction through the receptor, wherein the constant K _{The D} -ab binding of the compound-trimer-antibody complex is 1 nM or less (down to about 1 pM). Typically, the member of the TNF superfamily is TNFa, most preferably TNFa _s .

Typically, the binding affinity of the antibody to the complex is reduced by at least about 100-fold (affinity is improved by at least about 100-fold), preferably by about 200-fold, compared to the binding affinity of the compound in the absence of the TNF trimer and/or binding to the TNF trimer in the absence of connection.

The compound-trimer complex can bind to an antibody comprising the amino acid sequences described herein.

The compound or complex described herein can be used in the treatment and/or prevention of a pathological condition. Accordingly, a compound or complex is provided for practical use in a method of drug treatment of a human or animal body and a method of treatment comprising administering the compound or complex. The compound or complex can be used for the treatment of any therapeutic indication and/or for the preparation of a pharmaceutical composition described in this context.

Antibodies.

The invention describes antibodies that selectively bind to at least one compound-trimer complex, including at least one compound as described herein and a trimeric member of the TNF superfamily.

Typically, selective binding of an antibody to a compound-trimer complex is measured relative to antibody binding to a compound in the absence of a TNF superfamily member, or to a TNF superfamily member in the absence of a compound, or binding to other (different) compound-trimer complexes.

First, the invention describes an antibody that selectively binds to a complex comprising (i) a trimeric protein that is a member of the TNF superfamily, and (ii) a compound that is capable of binding to a trimeric protein that is a member of the TNF superfamily, thereby forming a complex compound -trimer binds to an essential TNF superfamily receptor and modulates trimer-induced signal transduction through the receptor. Typically, said antibody binds selectively to said complex compared to its binding to a member of the TNF superfamily in the absence of a compound, or to a compound in the absence of a member of the TNF superfamily.

- 10 045234

The compound may be any compound described above, including compounds (1)-(6) (or salts or solvates thereof). As will be discussed below, a member of the TNF superfamily can be any member of the superfamily, but typically TNFa. More specifically, TNFa is human TNFa, primarily soluble TNFa (TNFas). TNFas may be characterized by the sequences of SEQ ID NO: 35 or SEQ ID NO: 36, or may be variants of the sequences of SEQ ID NO: 35 or SEQ ID NO: 36. Such variants typically retain at least about 60, 70, 80, 90, 91, 92, 93, 94, or 95% identity to SEQ ID NO: 35 or SEQ ID NO: 36 (or even approximately 96, 97, 98, or 99% identity). In other words, such variants may retain about 60% to about 99% identity to SEQ ID NO: 35 or SEQ ID NO: 36, preferably about 80% to about 99% identity to SEQ ID NO: 35 or SEQ ID NO: 36 , more preferably about 90% to about 99% identity to the sequence of SEQ ID NO: 35 or SEQ ID NO: 36, and most preferably about 95% to about 99% identity to the sequence of SEQ ID NO: 35 or SEQ ID NO: 36. Variations are described in more detail below.

The term corresponding sequence means that TNFa may be characterized by the wild-type amino acid sequence of any known animal or human TNFa, especially human TNFa, for example, the sequence SEQ ID NO: 36. TNFa may include soluble TNFa (sTNFa) or membrane-bound TNFa or both types squirrel. Soluble homotrimeric TNFa (sTNF) is released from membrane-bound homotrimeric TNFa (mTNF) by proteolytic cleavage by TNFa-converting enzyme (TACE/ADAM17; although other proteinases can also release sTNF, such as ADAM10, ADAM19, matrix metalloproteinase 7 and proteinase 3, upon cleavage which produces the corresponding soluble TNFa sequences, which may be extended or shortened by 1, 2, 3, 4 or 5 amino acid residues compared to sTNF (such as the sequence SEQ ID NO: 36) obtained after cleavage by TACE enzyme) . Soluble trimeric sTNF, molecular weight 52 kDA, has the shape of a tetrahedron. The human sequence called mTNF is shown in SEQ ID NO: 34, and the human sequence called sTNF (the product of the TACE enzyme on SEQ ID NO: 34) is shown in SEQ ID NO: 36. Corresponding rat and mouse mTNFa sequences presented in the sequences SEQ ID NO: 32 and 33, respectively. Corresponding TNFa sequences from other animals (or known human sequence variants) can simply be combined with SEQ ID NO: 36 and assigned numbering similar to SEQ ID NO: 36 (used herein to number TNFa amino acid residues). For example, sequences from various animals can be found in the Uniprot database (www.uniprot.org), including human sequences P01375 and Q5STB3. Corresponding sTNFa sequences may include a C-terminal fragment of the mTNFa sequence (such as SEQ ID NO: 36) containing 157 amino acid residues, or may be shortened or extended by one, two or three amino acid residues (rat and mouse sequences include 156 amino acid residues). The corresponding sTNFa sequence may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid substitutions compared to the sequence SEQ ID NO: 36. The corresponding sTNFa sequence may have 80, 90, 95, 96, 97, 98, or 99% amino acid identity to SEQ ID NO: 36 over the entire length of SEQ ID NO: 36.

As discussed above, although the present disclosure primarily relates to the binding of antibodies to trimers of members of the TNF superfamily, antibodies may also bind to other forms of a member of the TNF superfamily. For illustration, the Examples section of the present invention shows that antibody CA185_0179 binds to trimeric TNF. However, as shown in FIG. 1 (crystal structure of antibody CA185_0179) bound to TNFa monomer in the presence of compound (1)), the antibody also binds to TNFa monomer. Without wishing to be bound by theory, it is believed that in the presence of the compound, the soluble domain of TNF retains its trimeric structure. However, subunits A and C move away from each other (and subunit C rotates), creating a pocket between the two subunits. Thus, although antibodies bind to disrupted trimers, there is also the possibility that antibodies can still bind if the monomers in the trimer structure are pulled apart.

Regarding subunits A and C, if you look at the crystal structure of the TNFa trimer from the outside, it takes on a pyramid/cone-like shape. If you look along the axis of the trimer, so that the N- and C-terminal residues of the monomer point towards you when you look from the flat end of the trimer. When the structure is disrupted by the compound, it is believed without being limited by theory that a pocket opens between the A and C subunits in which the antibodies bind to the trimer.

The arrangement of chains A, B or C can be determined by measuring the three distances between the three a- 11 045234 carbon atoms of three identical residues, for example P117, in each chain (residue G121 can also be used).

The three distances form a triangle, which is equilateral in the case of apoTNF but becomes distorted when bound to a compound. The shortest distance is observed between BC, and the longest distance is observed between AC (for example, AC = 13.8 A, AB = 12.3 A, BC = 10.2 A), and thus, when looking along the axis of the molecule, when The N/C-terminal residues point to you, then the longest distance is determined by chain C, then chains A in a counterclockwise direction, then chains B and C again in a counterclockwise direction.

Accordingly, antibodies are also provided that selectively bind to a complex comprising human TNFa and a compound selected from the group consisting of compounds (1) to (6) or salts or solvates thereof. TNFa is usually soluble TNFa (TNFas). TNFas may include the sequence SEQ ID NO: 35 or SEQ ID NO: 36, or variants thereof. Such variants may retain at least about 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 35 or SEQ ID NO: 36 (see above or the methods for identifying variants described below). TNFa may be trimeric.

The term antibody as used herein includes whole antibodies and any antigen-binding fragment (ie, antigen-binding portion) and individual chains thereof. Antibody means a glycoprotein comprising at least two heavy chains (H) and two light chains (L) linked by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain consists of a heavy chain variable region (abbreviated here as HCVR or VH) and a conserved heavy chain region. Each light chain consists of a light chain variable region (abbreviated here as LCVR or VL) and a light chain conserved region. The heavy and light chain variable regions contain a binding domain that interacts with the antigen. The V _H and V _L regions can be further subdivided into hypervariable regions, so-called complementarity determining regions (CDRs), interspersed with more conserved regions, so-called framework regions (FR).

Conserved regions of antibodies can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.

The antibody may be a monoclonal antibody or a polyclonal antibody and is typically a monoclonal antibody. The antibody may be a chimeric antibody, a CDR-grafted antibody, a nanoantibody, a human or humanized antibody, or an antigen-binding portion of any of these antibodies. To produce both types of antibodies, monoclonal and polyclonal, the experimental animals are usually non-human mammals such as goat, rabbit, rat or mouse, but the antibody can also be produced in other animal species.

Polyclonal antibodies can be produced by standard methods, such as immunization of a suitable animal with the antigen of interest. Blood can then be collected from the animal and the IgG fraction can be purified.

Antibodies raised against compound-trimer complexes can be obtained, if immunization of an animal is necessary, by administering the polypeptides to an animal, for example a non-human mammal, using known and standard protocols, see, for example, Handbook of Experimental Immunology, D.M. Weir (ed.), Vol 4, Blackwell Scientific Publishers, Oxford, England (1986). A variety of warm-blooded animals such as rabbits, mice, rats, sheep, cattle, camels or pigs can be immunized. However, the most suitable are mice, rabbits, pigs and rats.

Monoclonal antibodies can be produced by any method known in the art, such as the hybridoma method (Kohler & Milstein, Nature, 256:495-497 (1975)), the triome method, the human B cell hybridoma method (Kozbor et al., Immunology Today, 4:72 (1983)) and the EBV hybridoma method (Cole et al., Monoclonal Antibodies and Cancer Therapy, cc.77-96, Alan R Liss, Inc., (1985)).

Antibodies can also be produced by the antibody method from individual lymphocytes by cloning and expressing immunoglobulin variable region cDNA derived from individual lymphocytes selected to produce specific antibodies, for example, as described in Babcook, J. et al., Proc. Natl. Acad. Sci. USA 93(15): 7843-78481 (1996), WO92/02551, WO2004/051268 and WO2004/106377.

Antibodies can be produced using various phage display methods known in the art, including methods described in the following articles and applications: Brinkman et al., J. Immunol. Methods, 182: 41-50 (1995), Ames et al., J. Immunol. Methods, 184:177-186 (1995), Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994), Persic et al., Gene, 187 9-18 (1997), Burton et al., Advances in Immunology, 57:191-280 (1994) and WO 90/02809, WO 91/ 10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO 95/20401 and US 5698426, 5223409, 5403484, 5580717, 5427908, 5750753, 5821047, 5571698,

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5427908, 5516637, 5780225, 5658727, 5733743 and 5969108.

Whole human antibodies are antibodies in which all variable and conserved regions (if present) of both light and heavy chains originate from human antibodies, or are substantially identical to the sequences of human antibodies, but not necessarily the same antibody. Examples of whole human antibodies may include antibodies produced, for example, by the phage display methods described above, and antibodies produced in mice in which the murine immunoglobulin variable and optionally conserved region genes are replaced with corresponding human counterparts, for example, as generally described in the following patents: EP 0546073, US 5545806, US 5569825, US 5625126, US 5633425, US 5661016, US 5770429, EP 0438474 and EP 0463151.

In another embodiment, antibodies can be produced by a procedure comprising: immunizing a non-human mammal with an immunogen comprising a compound-trimer complex of a member of the TNF superfamily and a compound described herein, obtaining an antibody preparation from said mammal, obtaining monoclonal antibodies therefrom, that selectively recognize the complex, and screening a population of monoclonal antibodies that bind to a member of the TNF superfamily only in the presence of the compound.

Antibody molecules may include a complete antibody molecule containing full-length heavy and light chains or a fragment thereof or an antigen-binding portion. The term antigen-binding portion of an antibody means one or more antibody fragments that retain the ability to selectively bind an antigen. It has been found that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Antibodies and fragments and antigen-binding portions thereof may be, but are not limited to, Fab, modified Fab, Fab', modified Fab', F(ab') ₂ , Fv, individual antibody domains (e.g., VH or VL or VHH ), scFv, di-, tri- or tetravalent antibodies, Bis-scFv, diabodies, tri-bodies, tetrabodies and epitope-binding fragments of any of these parts of the antibody (see, for example, Holliger and Hudson, Nature Biotech. 23(9): 1126- 1136 (2005), Adair and Lawson, Drug Design Reviews - Online 2(3), 209-217 (2005)). Methods for designing and producing these antibody fragments are well known in the art (see, for example, Verma et al., Journal of Immunological Methods, 216, 165-181 (1998)). Other antibody fragments include Fab and Fab' fragments described in WO 2005/003169, WO 2005/003170 and WO 2005/003171, and Fab-dAb fragments described in WO 2009/040562. Multivalent antibodies may include multiple specificities or may be monospecific (see, for example, WO 92/22853 and WO 05/113605). These antibody fragments can be prepared using techniques known in the art, and the fragments can be screened using a method similar to that for intact antibody screening.

The domains of conserved regions of the antibody molecule, if present, can be selected based on the intended function of the antibody molecule, and especially the effector functions that may be required. For example, conserved region domains may include human IgA, IgD, IgE, IgG, or IgM domains. First of all, domains of the conserved region of IgG, especially the IgG1 and IgG3 isotypes, can be used if the antibody molecule is intended for therapeutic use and the effector functions of the antibody are required. Alternatively, the IgG2 and IgG4 isotypes may be used if the antibody molecule is intended for therapeutic purposes and the effector functions of the antibody are not required.

The antibody can be obtained, expressed, constructed or isolated by recombinant methods, such as (a) antibodies isolated from an animal (for example, a mouse) that is transgenic or transchromosomal for the genes of the immunoglobulins of interest, or from a hybridoma obtained from them, (b ) antibodies isolated from host cells transformed to express the antibody of interest, for example, from a transfectome, (c) antibodies isolated from a combinatorial library of recombinant antibodies, and (d) antibodies produced, expressed, engineered or isolated by any other method that involves the splicing of immunoglobulin gene sequences to form other DNA sequences.

The antibody may be a human antibody or a humanized antibody. The term human antibody as used herein is intended to include antibodies containing variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Moreover, if the antibody contains a conserved region, then the conserved region is derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues that are not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-directed mutagenesis in vitro or somatic mutation in vivo). However, the term human antibody as used in this context is not intended to include antibodies in which the CDR sequences are derived from the germ line of other mammalian species, such as mouse, that are grafted onto human framework sequences.

Such a human antibody may be a human monoclonal antibody. Such a human monoclonal antibody can be produced in a hybridoma that includes a B cell derived from a transgenic non-human animal, for example a transgenic mouse, comprising a genome containing a human heavy chain transgene and a human light chain transgene hybridized into immortal cell.

Human antibodies can be obtained by in vitro immunization of human lymphocytes followed by transformation of the lymphocytes with the Epstein-Barr virus.

The term human antibody derivatives means any modified form of a human antibody, such as a conjugate of a human antibody and another agent or antibody.

The term humanized antibody is intended to mean CDR-grafted antibody molecules in which the CDR sequences are derived from the germ line of another mammalian species, such as a mouse, that are grafted onto human framework sequences. Additional modifications to the framework region can be made to human framework sequences.

As used herein, the term CDR-grafted antibody molecules means an antibody molecule in which the heavy and/or light chain contains one or more CDR fragments (including, if necessary, one or more modified CDR fragments) from a donor antibody (for example, a mouse monoclonal antibody or rats) grafted onto the variable framework region of the heavy and/or light chain of an acceptor antibody (eg, a human antibody). See review by Vaughan et al., Nature Biotechnology, 16, 535-539, (1998). In one embodiment, instead of transfecting the entire CDR fragment, only one or more specificity-determining residues from any CDR fragment described herein above are transfected into the human antibody framework fragment (see, for example, Kashmiri et al., Methods, 36, 25- 34 (2005)). In one embodiment, only the specificity-determining residues from one or more of the CDR fragments described above are transfected into the human antibody framework region. In another embodiment, only the specificity-determining residues from each CDR region described herein above are transfected into the human antibody framework region.

If CDR fragments or specificity-determining residues are grafted, any appropriate acceptor variable region framework sequence may be used, taking into account the class/type of donor antibody from which the CDRs are derived, including mouse, primate and human framework regions. Accordingly, the CDR-grafted antibody contains a variable domain including human acceptor framework regions as well as one or more CDRs or specificity-determining residues described above. Thus, a neutralizing CDR-grafted antibody is provided in which the variable domain includes human acceptor framework regions and a non-human donor animal CDR.

Examples of human framework regions include KOL, NEWM, REI, EU, TUR, TEI, LAY, and POM (Kabat et al., supra). For example, the KOL and NEWM regions can be used for the heavy chain, the REI region can be used for the light chain, and the EU, LAY, and POM regions can be used for both the heavy and light chains. Alternatively, human germline sequences can be used, which can be found, for example, at http://www.vbase2.org/ (Retter et al., Nucl. Acids Res. 33 (supplement 1), D671 -D674 (2005)).

In the case of a CDR-grafted human antibody, the acceptor heavy and light chains do not necessarily need to be derived from the same antibody, and may optionally include mixed chains containing framework regions from different chains.

Likewise, a CDR-grafted antibody does not need to use acceptor framework regions with the exact same sequence as the acceptor antibody. For example, atypical residues can be replaced by more common residues for an acceptor chain of that class or type. Alternatively, selected residues in the acceptor framework regions can be replaced by residues that correspond to residues located at the same positions in the donor antibody (Reichmann et al., Nature, 332, 323-324 (1998)). Such changes should be kept to a minimum to maintain the affinity of the donor antibody. A protocol for selecting residues in the acceptor framework regions required for replacement is presented in WO 91/09967.

One skilled in the art will recognize that antibodies can undergo various post-translational modifications. In most cases, the type and extent of these modifications depend on the host cell line used to express the antibody, as well as the culture conditions. Such modifications may include variations in glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization, and asparagine deamination. The most common modifications include the loss of a C-terminal basic amino acid residue (such as lysine or arginine) due to the action of carboxypeptidases (as described in Harris RJ., Journal of Chromatography 705:129-134 (1995)).

In one embodiment, the antibody heavy chain includes a CH1 domain and the antibody light chain includes a CL, either kappa or lambda domain.

Biological molecules, such as antibodies or fragments, contain acidic and/or basic functional groups that give the molecule a net positive or negative

- 14 045234 charge. The amount of total charge observed depends on the complete amino acid sequence of the molecule, the local arrangement of charged groups in the three-dimensional structure, and the conditions surrounding the molecule. Isoelectric point (IEP) refers to the pH at which a particular molecule or surface does not contain any electrical charge. In one embodiment, the IET of the antibody or fragment is at least 7. In another embodiment, the IET of the antibody or fragment is at least 8, such as 8.5, 8.6, 8.7, 8.8, or 9. In yet another embodiment The IET of an antibody is 8. The **ExPASY programs http://www.expasy.ch/tools/pi_tool.html can be used to predict the IET value of an antibody or fragment (see Walker, The Proteomics Protocols Handbook, Humana Press 571-607 ( 2005)).

The antibody may include at least one, two, or all three heavy chain CDR sequences of SEQ ID NOs: 4-6 (HCDR1/HCDR2/HCDR3, respectively). The sequences of the antibody CA185_01974 described in the Examples section include the sequences HCDR1/HCDR2/HCDR3.

Moreover, the antibody may include two or all three light chain CDR sequences of SEQ ID NOs: 1-3 (LCDR1/LCDR2/LCDR3, respectively). Such sequences of the antibody CA185_01974 described in the Examples section include the sequences LCDR1/LCDR2/LCDR3.

Antibodies preferably include at least HCDR3, sequence SEQ ID NO: 6.

Typically, the antibody comprises at least one of the heavy chain CDR sequences SEQ ID NOs: 4-6 and at least one of the light chain CDR sequences SEQ ID NOs: 1-3. The antibody may include at least two heavy chain CDR sequences selected from SEQ ID NOs: 4-6 and at least two CDR sequences selected from SEQ ID NOs: 1-3. Typically, the antibody includes all three heavy chain CDR sequences SEQ ID NOs: 4-6 (HCDR1/HCDR2/HCDR3, respectively) and all three light chain CDR sequences SEQ ID NOs: 1-3 (LCDR1/LCDR2/LCDR3, respectively). Antibodies can be chimeric, humanized, or human antibodies.

The antibody may also include at least one, at least two, or all three heavy chain CDR sequences of SEQ ID NOs: 19-21 (HCDR1/HCDR2/HCDR3, respectively). The sequences of the antibody CA185_01979 described in the Examples section include the sequences HCDR1/HCDR2/HCDR3.

Typically the antibody includes the HCDR3 sequence of SEQ ID NO: 21.

The antibody may also include at least one, at least two, or all three light chain CDR sequences of SEQ ID NOs: 1, 17, 18 (LCDR1/LCDR2/LCDR3, respectively). The sequences of the antibody CA185_01979 described in the Examples section include the sequences LCDR1/LCDR2/LCDR3.

Typically, the antibody includes at least one heavy chain CDR sequence selected from SEQ ID NOs: 19-21, and at least one light chain CDR sequence selected from SEQ ID NOs: 1, 17, 18. The antibody may include at least two heavy chain CDR sequences selected from SEQ ID NOs: 19-21, and at least two light chain CDR sequences selected from SEQ ID NOs: 1, 17, 18. The antibody typically includes all three heavy chain CDR sequences of SEQ ID NOs : 19-21 (HCDR1/HCDR2/HCDR3, respectively) and all three light chain CDR sequences SEQ ID NOs: 1, 17, 18 (LCDR1/LCDR2/LCDR3, respectively). Antibodies can be chimeric, humanized, or human antibodies.

The antibodies may include any combination of the CDR sequences of antibody CA185_01974 and antibody CA185_01979. First, the antibody may include at least one HCDR sequence selected from SEQ ID NOs: 4-6 and 19-21 and/or at least one LCDR sequence selected from SEQ ID NOs: 1-3, 17 and 18.

The antibody may include:

HCDR1 selected from SEQ ID NOs: 4 and 19, and/or

HCDR2 selected from SEQ ID NOs: 5 and 20, and/or

HCDR3 selected from SEQ ID NOs: 6 and 21; and/or

LCDR1 SEQ ID NO: 1, and/or

LCDR2 selected from SEQ ID NOs: 2 and 17, and/or

LCDR3 selected from SEQ ID NOs: 3 and 18.

The antibody may also include a heavy chain variable region (HCVR) sequence of SEQ ID NO: 8 (HCVR region of antibody CA185_01974). The antibody may also include a light chain variable region (LCVR) sequence of SEQ ID NO: 7 (antibody LCVR region CA185_01974). Preferably, the antibody of the invention comprises a heavy chain variable region of SEQ ID NO: 8 and a light chain variable region of SEQ ID NO: 7.

The antibody may also include a heavy chain variable region (HCVR) sequence of SEQ ID NO: 23 (HCVR antibody region CA185_01979). The antibody may comprise a light chain variable region (LCVR) sequence of SEQ ID NO: 22 (antibody LCVR region CA185_01979). The antibody preferably comprises a heavy chain variable region of SEQ ID NO: 23 and a light chain variable region of SEQ ID NO: 22.

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Antibodies may include a combination of antibody heavy and light chain variable regions

CA185_01974 and CA185_01979. In other words, the antibody may comprise a heavy chain variable region of SEQ ID NO: 8 or 23, and/or a light chain variable region of SEQ ID NO: 7 or 22.

The antibody may include a heavy chain (H chain) sequence, SEQ ID NO: 12 (CA185_01974 mIgG1) or 13 (CA185_01974 mFab (no hinge)). The antibody may comprise a light chain (L chain) sequence, SEQ ID NO: 11 (kappa light chain CA185_01974). The antibody typically comprises a heavy chain sequence of SEQ ID NO: 12/13 and a light chain sequence of SEQ ID NO: 11. Antibodies can be chimeric, humanized, or human antibodies.

Antibodies may include the heavy chain sequence of SEQ ID NO: 27 (CA185_01979 mIgG1) or 28 (CA185_01979 mFab (no hinge)). Antibodies may include the light chain sequence of SEQ ID NO: 26 (CA185_01979 kappa light chain). Generally, the antibody comprises a heavy chain sequence of SEQ ID NO: 27/28 and a light chain sequence of SEQ ID NO: 26. Antibodies can be chimeric, humanized, or human antibodies. Again, sequences from antibodies CA185_01974 and CA185_01979 can be combined.

In another embodiment, the antibody may be a variant of or include a variant of one of the specific sequences listed above. For example, a variant may include a substitution, deletion, or insertion of any of the sequences listed above.

An antibody variant may include 1, 2, 3, 4, 5, up to 10, up to 20 or more (typically up to a maximum of 50) substitutions and/or deletions of amino acid residues in the specific sequences listed above. The term deletion can mean the deletion of individual amino acid residues, the deletion of small groups of amino acid residues, such as 2, 3, 4 or 5 amino acid residues, or the deletion of large regions of amino acid residues, such as the deletion of specific amino acid domains or other features. Substitution options typically involve replacing one or more amino acid residues with the same number of amino acid residues, provided that only conserved amino acid residues are replaced. For example, an amino acid can be replaced by another amino acid with similar properties, such as another basic amino acid, another acidic amino acid, another neutral amino acid, another charged acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid, or another aliphatic amino acid. Some properties of the most common amino acids that can be selected for suitable substitutions are presented in the following table:

Ala aliphatic, hydrophobic, neutral Met hydrophobic, neutral Cys polar, hydrophobic, neutral Asn polar, hydrophilic, neutral Asp polar, hydrophilic, neutral (-) Pro Hydrophobic, neutral Glu polar, hydrophilic, charged (-) Gln polar, hydrophilic, neutral Phe aromatic, hydrophobic, neutral Arg polar, hydrophilic, charged (+) Gly aliphatic, neutral Ser polar, hydrophilic, neutral His aromatic, polar, hydrophilic, charged (+) Thr polar, hydrophilic, neutral lie aliphatic, hydrophobic, neutral Val aliphatic, hydrophobic, neutral Lys polar, hydrophilic, charged(+) Trp aromatic, hydrophobic, neutral Leu aliphatic, hydrophobic, neutral Tyr aromatic, polar, hydrophobic

Derivatives or variants generally include substitutions in which the naturally occurring amino acid is replaced by its structural analogue. The amino acids used in the sequences can be changed or modified, for example by including a tag, as long as there is no significant negative effect on the function of the antibody.

The derivatives and variants described above can be obtained by synthesizing the antibody or modifying it after production, or the antibody can be produced in recombinant form using known methods of site-directed mutagenesis, non-specific mutagenesis, or enzymatic digestion and/or nucleic acid ligation.

- 16 045234

Antibody variants may include an amino acid sequence that has an amino acid identity of greater than about 60%, preferably greater than 70%, such as 75 or 80%, preferably greater than about 85%, such as greater than about 90 or 95%, to amino acid sequences described in this context (primarily the HCVR/LCVR sequences and the H and L chain sequences). Moreover, the antibody may be a variant that has amino acid identity of greater than about 60%, or greater than about 70%, such as about 75% or 80%, typically greater than about 85%, such as greater than about 90 or 95% compared to the HCVR/LCVR amino acid sequences and the H and L chain sequences described herein, while retaining the complete CDR regions described for these sequences. Variants may retain at least about 90, 91, 92, 93, 94%, 95, 96, 97, 98, or 99% identity compared to the HCVR/LCVR sequences and the H and L chain sequences described herein (in some In some cases, the complete CDR areas are retained).

The variants typically retain about 60% to about 99% identity, about 80% to about 99% identity, about 90% to about 99% identity, or about 95% to about 99% identity. This level of amino acid identity may be observed along the entire length of the corresponding SEQ ID NO or a portion of the sequence, such as 20, 30, 50, 75, 100, 150, 200 or more amino acid residues, depending on the size of the overall length of the polypeptide.

With regard to amino acid sequences, the term sequence identity refers to sequences for which the value of identity is established by evaluation using the ClustalW program (Thompson et al., 1994, supra) with the following parameters:

Pairwise alignment parameters: Method: accurate, Matrix: RAM, Gap open penalty: 10.00, Gap extension penalty: 0.10.

Multiple alignment parameters: Matrix: RAM, Gap open penalty: 10.00, % identity for delay: 30, Penalize end gaps: on, Gap separation distance: 0, Negative matrix: by (no), Gap extension penalty: 0.20, Residue-specific gap penalties (penalty for missing ( gap) of a specific amino acid residue): on (on), Hydrophilic gap penalties (penalty for a hydrophilic gap): on (on), Hydrophilic residues (hydrophilic residues): GPSNDQEKR. The sequence identity for a particular residue is assumed to include identical residues that have simply been modified.

The present invention also provides an isolated DNA sequence encoding the variable regions of the heavy and/or light chains of an antibody molecule. Thus, the present invention describes an isolated DNA sequence of SEQ ID NO: 10 encoding the heavy chain variable region of SEQ ID NO: 8, as well as an isolated DNA sequence SEQ ID NO: 9 encoding the light chain variable region of SEQ ID NO: 7.

Also described is the isolated DNA sequence SEQ ID NO: 25 encoding the heavy chain variable region of SEQ ID NO: 23 and the isolated DNA sequence SEQ ID NO: 24 encoding the light chain variable region SEQ ID NO: 22.

In addition, an isolated DNA sequence of SEQ ID NO: 15 or 16 encoding the heavy chains of SEQ ID NOs: 12 and 13, respectively, as well as an isolated DNA sequence of SEQ ID NO: 14 encoding the light chain of SEQ ID NO: 11 are disclosed.

The invention describes an isolated DNA sequence of SEQ ID NO: 30 or 31, encoding the heavy chains of SEQ ID NO: 27 and 28, respectively, as well as an isolated DNA sequence of SEQ ID NO: 29, encoding the light chain of SEQ ID NO: 26.

In another embodiment, a suitable polynucleotide sequence may be a variant of one of these specific polynucleotide sequences. For example, a variant may be a substitution, deletion, or insertion variant of any of the nucleotide sequences listed above. The polynucleotide variant may include 1, 2, 3, 4, 5, up to 10, up to 20, up to 30, up to 40, up to 50, up to 75 or more nucleotide substitutions and/or deletions in the sequences specified in list of sequences. Basically, the variant includes 1-20, 150, 1-75 or 1-100 substitutions and/or deletions.

The homology of suitable variants to a polynucleotide of any one of the nucleotide sequences described herein can be at least about 70%, typically at least about 80 or 90%, and most preferably about 95, 97 or 99%. The identity of the variants may be maintained at a level of at least about 90%, 91, 92, 93, 94, 95, 96, 97, 98, or 99%. The identity of the variants may be maintained at a level of about 60% to about 99%, about 80% to about 99%, about 90% to about 99%, or about 95% to about 99%. Homology and identity at this level generally relate to at least the coding regions of polynucleotides. Race methods

- 17 045234 four homologies are widely known in the art, and it is obvious to those skilled in the art that homology is calculated based on nucleotide identity. Such homology may refer to a region comprising at least about 15, at least about 30, such as at least about 40, 60, 100, 200, or more contiguous nucleotides (depending on length). Such homology may extend to the entire length of the unmodified polynucleotide sequence.

Methods for calculating polynucleotide homology or identity are known in the art. For example, the UWGCG software package offers the BESTFIT program, which can be used to calculate homology (eg, using default parameters) (see Devereux et al., Nucleic Acids Research 12, cc. 387-395 (1984)).

The PILEUP and BLAST algorithms can also be used to calculate homology and sequence alignment (usually using default parameters), for example, as described in Altschul S.F., J Mol Evol 36:290-300 (1993), Altschul S.F. et al., J Mol Biol 215:403-10 (1990).

BLAST analysis software is publicly available from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying a High Similarity Score (HSP) sequence pair by identifying short words of length W in a query sequence that match or meet a certain threshold positive value T when aligning a word of the same length in a database sequence. T means the threshold level of positive values of the neighboring word (Altschul et al., see above). Such matches of the original neighboring words trigger a search for HPSs containing them. Word matches are propagated in both directions along each sequence as long as the cumulative alignment score can be increased. The propagation of word matches in each direction stops when the cumulative alignment score is reduced to zero or below by the accumulation of one or more negatively scored residue alignments, or when the end of one of the sequences is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of alignment. BLAST defaults to word length (W) 11, BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919 ((1992)), number of alignments (B) 50, expectation (E) 10, M=5, N=4, and compare both circuits.

The BLAST program can perform statistical analysis of the similarity of two sequences, see, for example, Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993). One measure of similarity determined by the BLAST algorithm is the lowest cumulative probability (P(N)), which estimates the probability of a random match between two nucleotide or amino acid sequences. For example, a sequence may be considered similar to another sequence if the lowest overall probability when comparing the first sequence to a second sequence is less than about 1, typically less than about 0.1, preferably less than about 0.01, and most preferably less than about 0.001. For example, the lowest overall probability may range from about 1 to about 0.001, in most cases from about 0.01 to about 0.001.

A homologous sequence may differ from the sequence in the corresponding polynucleotide by the presence of less than about 3, 5, 10, 15, 20 or more mutations (in each case, substitutions, deletions or insertions). For example, a homologous sequence may differ in the presence of 3-50 mutations, in most cases 3-20 mutations. These mutations can be detected in a region comprising at least 30, for example at least 40, 60 or 100 or more contiguous nucleotides in homologous sequence.

A sequence variant may differ from the specific sequences listed in the sequence listing due to redundancy in the genetic code. The DNA code contains three primary nucleotide residues (A, T, C and G) and uses these bases to read three-letter codons, which represent the amino acids of proteins encoded by the body's genes. The linear sequence of codons along the DNA molecule is translated into the linear amino acid sequence of the protein(s) encoded by these genes. The code is highly degenerate and includes 61 codons encoding 20 natural amino acids and 3 codons representing stop signals. Thus, most amino acids are encoded by more than one codon—indeed, several amino acids are encoded by four or more different codons. Therefore, a polynucleotide variant may encode the same polypeptide sequence as another polynucleotide, but have a different nucleotide sequence by using different codons to encode the same amino acids.

The DNA sequence may include synthetic DNA, such as chemically processed DNA, cDNA, genomic DNA, or any combination thereof.

DNA sequences encoding the antibody molecule can be obtained by methods that are well known to one skilled in the art. For example, DNA sequences encoding part or all of the heavy and light chain sequences of an antibody can be synthesized, if desired, from specific DNA sequences or based on corresponding amino acid sequences.

Standard methods by which vectors can be constructed, transfection methods, and culture methods are well known to one skilled in the art. These methods are described in the book Current Protocols in Molecular Biology, ed. F. M. Ausubel, ed. Wiley Interscience, New York (1999) and in the Maniatis Manual, ed. Cold Spring Harbor.

The invention also describes a host cell that contains one or more cloning or expression vectors containing one or more DNA sequences encoding an antibody. Any suitable host cell/vector system can be used to express the DNA sequences encoding the antibody. Bacterial systems, such as E. coli and other microbial systems, can be used, and eukaryotic host cell-based expression systems, such as mammalian cells, can also be used. Suitable mammalian host cells include CHO cells, myeloma cells or hybridoma cells.

The invention also describes a method for producing an antibody, including culturing host cells containing the vector under conditions suitable for protein expression involving DNA encoding the antibody, as well as isolating the antibody.

Screening methods, as described herein, can be used to identify suitable antibodies that are capable of binding to the compound-trimer complex. Thus, the screening methods described in this context can be used to test the antibodies of interest.

The assessment of antibody binding to the compound-trimer complex can be carried out, for example, using a standard ELISA or Western blotting. The ELISA method can also be used to screen for hybridomas that are characterized by positive reactivity for the target protein. The selectivity of antibody binding can also be determined by monitoring the binding of antibodies to cells expressing the target protein, for example, using flow cytometry. Thus, the screening method may include the step of identifying antibodies that are capable of binding to the compound-trimer complex by ELISA or Western blotting or flow cytometry.

Antibodies selectively (or specifically) recognize at least one compound-trimer complex, i.e. epitopes in the compound-trimer complex. An antibody or other compound selectively binds or selectively recognizes a protein if it binds preferentially to, or has high affinity for, a protein for which it is selective but has little or no binding to other proteins. The selectivity of an antibody for a target compound-trimer complex can in turn be assessed by the binding of the antibody to complexes of other similar trimer compounds as described above, or by the absence of such binding, or by the ability of the antibody to distinguish one complex from another.

The antibody may specifically (or selectively) bind to compound-trimer complexes containing trimeric forms of one or more members of the TNF superfamily. For example, the antibody can bind to compound-trimer complexes containing TNFa, compound-trimer complexes containing TNFe, and compound-trimer complexes containing CD40L. In another embodiment, the antibody may specifically (or selectively) bind to compound-trimer complexes containing only one member of the TNF superfamily, but not to compound-trimer complexes containing any other members of the TNF superfamily. For example, the antibody may bind to compound-trimer complexes containing TNFa, but not to compound-trimer complexes containing TNFe or compound-trimer complexes containing CD40L. The antibody may specifically (or selectively) bind to compound-trimer complexes containing up to two, three, four, or up to all members of the TNF superfamily.

It should be understood that the term specifically (or selectively) means that the antibody binds to the compound-trimer complexes of interest and does not exhibit significant cross-reactivity with any other compound, which may include the compounds of interest in the absence of a TNF superfamily trimer or trimers of TNF superfamily members in absence of the test compound. Cross-reactivity can be assessed by any method described in this context. Cross-reactivity of an antibody against a compound-trimer complex with another compound other than the compound-trimer complex can be considered significant if the degree of binding of the antibody to the other compound is increased by at least about 5, 10, 15, 20, 25, 30. 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 100% compared to binding to the test compound-trimer complex. The degree of binding of an antibody that specifically (or selectively) binds to a compound-trimer complex to another compound may be less than about 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30. 25 or 20% compared to the degree of binding to the compound-trimer complex.

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The extent of binding of an antibody to another compound may be less than about 20%, less than about 15%, less than about 10%, or less than about 5%, less than about 2%, or less than about 1% compared to the extent of its binding to the compound-trimer complex. The antibody specifically (or selectively) binds to the compound-trimer complex compared to (i) the trimeric form of the TNF superfamily member in the absence of the compound and/or (ii) the compound in the absence of the trimer of the TNF superfamily member.

The rate at which the antibody binds to the compound-trimer complex is in this context called the rate of association or on-rate, k _on-a b, and the rate at which the antibody dissociates, i.e. is separated from the associate with the compound-trimer complex, called in this context the dissociation rate or off-rate, k _off _ _ab . The symbol ''K _D . _ab '', as used in this context, denotes the binding affinity (dissociation constant) of the antibody to the compound-trimer complex. K _D . _ab means koff.ab/kon.ab. Antibodies may have low on-rate values, which can be measured in minutes based on mass spectrometric analysis of the compound-trimer complex and the intensity of the peak corresponding to the antibody. K _D values. _ab for an antibody can be assessed by repeating this measurement at different ratios of antibody/compound-trimer complex.

K _D values. _ab antibodies when bound to the compound-trimer complex can be reduced by at least approximately 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold , 70 times, 80 times, 90 times, 100 times, 200 times, 300 times or 400 times or even more compared to the value of K _D . _ab antibodies upon binding to a trimeric member of the TNF superfamily in the absence of a compound and/or K _D value. _ab antibodies upon binding to a compound in the absence of a trimeric member of the TNF superfamily. K _D value. _ab of an antibody when bound to a compound-trimer complex may be reduced by at least about 10-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold compared to the K _D value. _ab trimer of the TNF superfamily upon binding to the TNF superfamily receptor in the absence of the test compound, i.e. the binding affinity of antibodies to the compound-trimer complex is typically increased by at least about 10-fold, preferably at least about 100-fold, more preferably at least about 200-fold, even more preferably at least about 300-fold compared to the binding affinity of the antibody to a trimeric member of the TNF superfamily in the absence of a compound and/or the binding affinity of the antibody to a compound in the absence of a trimeric member of the TNF superfamily.

Binding affinity can be expressed as a binding affinity constant ( _KD.ab ) in any suitable units such as µM, nM or pM _. The smaller the value of K _D . _ab , the greater the binding affinity of the antibody to the compound-trimer complex.

K _D value. _ab antibodies when bound to the compound-trimer complex may be reduced by at least approximately 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold , 70 times, 80 times, 90 times, 100 times or more compared to the value of K _D . _ab antibodies upon binding to a trimeric member of the TNF superfamily in the absence of a compound and/or K _D value. _ab antibodies upon binding to a compound in the absence of a trimeric member of the TNF superfamily.

Decrease in K _D value. _{ab of} antibody binding to the compound-trimer complex compared to the K _D value. _ab binding of an antibody to a trimeric member of the TNF superfamily in the absence of a compound and/or a K _D value. _ab binding of an antibody to a compound in the absence of a trimeric member of the TNF superfamily may result from an increase in the rate of op-rate (kon.ab) of binding of an antibody to a compound-trimer complex compared to binding of an antibody to a trimeric member of the TNF superfamily in the absence of a compound and/or with binding antibodies to a compound in the absence of a trimeric member of the TNF superfamily, and/or may result from a decrease in off-rate (koff.ab) compared with binding of an antibody to a trimeric member of the TNF superfamily in the absence of a compound and/or with binding of an antibody to a compound in the absence trimeric member of the TNF superfamily.

The rate of on-rate (k _on . _ab ) binding of an antibody to a compound-trimer complex is generally increased relative to the rate of on-rate binding of an antibody to a trimeric member of the TNF superfamily in the absence of a compound and/or binding of an antibody to a compound in the absence of a trimeric member of the TNF superfamily. The rate of off-rate (koff.ab) binding of an antibody to a compound-trimer complex is generally reduced compared to the rate of off-rate binding of an antibody to a trimeric member of the TNF superfamily in the absence of the compound and/or binding of an antibody to a compound in the absence of a trimeric member of the TNF superfamily. Most often, the on-rate (kon.ab) rate of antibody binding to the compound-trimer complex is increased, and the off-rate (koff.ab) rate of antibody binding to the compound-trimer complex is reduced compared to antibody binding to a trimeric member of the TNF superfamily in the absence of compound and /or when an antibody binds to a compound in the absence of a trimeric member of the TNF superfamily.

The kon.ab value of antibody binding to the compound-trimer complex can be increased by at least about 1.5-fold or at least two-fold, and typically at least three-fold, compared to the kon.ab value of antibody binding to the trimer member TNF superfamily absent

- 20 045234 the binding and/or binding of an antibody to a compound in the absence of a trimeric member of the TNF superfamily, and/or the ko _ff-a b value of binding of an antibody to a compound-trimer complex may be reduced by at least about a factor of two, at least about a factor of 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times , at least about 80-fold, more preferably at least about 90-fold, compared to the ko _ff _ _ab value of antibody binding to a trimeric TNF superfamily member in the absence of a compound and/or antibody binding to a compound in the absence of a trimeric TNF superfamily member.

The values of k _on _ _ab , ko _ff _ _ab and K _D _ _ab can be determined by any suitable method, for example, surface plasmon resonance, mass spectrometry, and isothermal calorimetry.

The K _{D_ab} value of antibody binding to the compound-trimer complex may be 1 nM, 900 pM, 700 pM, 500 pM, 100 pM, 10 pM or less (typically down to about 1 pM ₎ . The antibodies described preferably bind to the compound-trimer complexes of the invention with high affinity, for example, in the picomolar range. The K _{D_ab} value of antibody binding to the compound-trimer complex may be 1 nM or less, 900 pM or less, 700 pM or less, 500 pM or less, 400 pM or less, 300 pM or less, 200 pM _or less, 100 pM or less, 90 pM or less, 80 pM or less, 70 pM or less, 60 pM or less, 50 pM or less, 40 pM or less, 30 pM or less, 20 pM or less, 10 pM or less (again , up to approximately 1 pM).

Once suitable antibodies have been identified and selected, the amino acid sequence of the antibodies can be determined by methods known to one skilled in the art. Genes encoding the amino acid sequence of antibodies can be cloned using degenerate primers. Recombinant antibodies can be produced using standard methods.

Antibodies may compete for binding to TNFa or bind to the same epitopes as described above for the H chain/L chain, HCVR/LCVR or CDR sequences. First, antibodies may compete for binding to TNFa or bind to the same epitopes as antibodies containing the sequence combination HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3, SEQ ID NO: 4/5/6/1/2/ 3 or SEQ ID NO: 19/20/21/1/17/18. Antibodies may compete for binding to TNFa or bind to the same epitopes as antibodies containing the sequence pair, HCVR and LCVR, SEQ ID NO: 8/7 or SEQ ID NO: 23/22.

The term epitope refers to the region of an antigen to which an antibody binds. Epitopes may be structural or functional epitopes. Functional epitopes are usually a subset of structural epitopes and contain amino acid residues that directly influence the affinity of the interaction. The epitopes may be conformational epitopes, i.e. consisting of non-linear amino acids (i.e. those that are not located directly next to each other). In certain embodiments, epitopes may include determinants that are reactive surface moieties of molecules, such as amino acids, saccharide side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments may have specific three-dimensional structural characteristics and/or specific charge characteristics.

The binding of an antibody to the same epitope or competition for binding with a reference antibody can be readily determined using standard techniques known in the art. For example, to determine the binding of a test antibody to the same epitope as a reference antibody, the binding of the reference antibody to a protein or peptide under saturation conditions is assessed. The ability of the test antibody to bind to the protein or peptide is then assessed. If the test antibody is able to bind to a protein or peptide after binding to the reference antibody under saturated conditions, then it can be concluded that the test antibody binds to a different epitope than the reference antibody. On the other hand, if the test antibody is unable to bind to a protein or peptide after binding to the reference antibody under saturated conditions, then the test antibody may bind to the same epitope as the reference antibody of the invention.

To assess the competitive binding of an antibody to a reference antibody, the binding procedure described above is carried out in two versions. In the first embodiment, the reference antibody is bound to the protein/peptide under saturation conditions, followed by assessment of the ability of the test antibody to bind to the specified protein/peptide. In the second option, the test antibody is bound to the protein/peptide under saturation conditions, followed by assessment of the ability of the reference antibody to bind to the specified protein/peptide. If in both variants only the first (saturated conditions) antibody is able to bind to the protein/peptide, then we can conclude that the test antibody and the reference antibody compete for binding to the protein/peptide. It will be apparent to one skilled in the art that an antibody that competes for antigen binding with a reference antibody does not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody as a result of binding

- 21 045234 names with an overlapping or adjacent epitope.

Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) the other from binding to the antigen. That is, a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits the binding of another by at least 50, 75, 90, or even 99% according to competitive binding assays (see, for example, Junghans et al. ., Cancer Res., 50, 1495-1502 (1990). In another embodiment, two antibodies bind to the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies bind to overlapping epitopes if certain amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

An additional standard experiment (e.g., peptide mutation and binding assay) can then be performed to confirm that the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody, or that steric blocking (or other phenomenon) is responsible for the observed lack of binding. Such experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available to one skilled in the art.

Antibodies can be used to identify compounds as described herein. Antibodies can also be used as biomarkers of target binding. A target binding biomarker can be used to detect binding, i.e. binding of the ligand to the target under study. In this case, antibodies bind only to complexes of compounds with trimeric forms of members of the TNF superfamily. Thus, if an antibody is able to bind to a compound-trimer complex, this indicates binding of the ligand to the target of interest (a trimer of a member of the TNF superfamily). Antibodies can be modified by introducing a detectable marker, as described in this context. Thus, the interaction of the compound with the target, i.e. with a target member of the TNF superfamily can be detected using such antibodies.

The use of antibodies as biomarkers of target binding may find use in clinical and preclinical trials in which a sample is obtained from a subject undergoing treatment according to the present invention. A sample obtained from a subject can be treated with antibodies to evaluate the binding of a compound used to treat the subject to a target member of the TNF superfamily. The sample obtained from the subject may be any suitable tissue or fluid, such as blood, blood plasma, or urine. The subject may be a mammal, usually a human.

Accordingly, the use of an antibody as a target binding biomarker is provided to detect a compound-trimer complex containing a trimeric protein that is a member of the TNF superfamily, as well as a compound that is capable of binding to a trimeric protein that is a member of the TNF superfamily, thereby making the compound-complex complex The trimer binds to an essential TNF superfamily receptor and modulates trimer-induced signal transduction through the receptor in a subject sample. The superfamily member is preferably TNFa and/or the modulation is antagonism of signaling through the TNFR1 receptor.

In addition, a method is provided for detecting target binding of a compound with a trimeric member of the TNF superfamily, whereby the compound-trimer complex binds to the desired receptor and modulates signal transduction induced by the trimer through the receptor, the method comprising:

(a) obtaining a sample from a subject who has been administered said compound, (b) contacting said antibody with said sample and said control sample, whereby antibodies are detected, (c) determining the amount of said detectable antibody bound with said sample and said control sample, wherein the higher the level of binding of said detectable antibody to said sample, compared with the level of binding of said detectable antibody to said control sample, indicates binding of said compound to the target, said trimeric member of the TNF superfamily.

Methods for detecting antibodies, as well as determining the amount of antibodies bound to a target, are well known in the art. Antibodies typically include tags. These labels include enzymes, biotin/streptavidin, fluorescent proteins and fluorescent dyes.

The binding of antibodies to the target can be measured, for example, by an immunochemical assay. Immunochemical analysis includes Western blotting, ELISA, immunofluorescence analysis, immunohistochemical analysis, and flow cytometry. Any suitable technique can be used to assess antibody binding to a member of the TNF superfamily.

In the method described above, the binding of a detectable antibody to a sample obtained from a subject administered the compound is compared with the binding of the antibody to a control sample.

- 22 045234

The control sample can be any suitable sample. The control sample is usually a negative control where the antibody binds to a member of the TNF superfamily in the absence of the compound. For example, a sample may be obtained from a patient prior to administration of the compound. Controls can also be obtained from samples used in previously conducted analyses, for example, selected from a number of samples from different subjects who were not administered the compound. Data from samples collected from 5, 10, 20, 50, or 100 subjects can be used to determine the reference value. The reference value may be the average or range of all values obtained.

The experiments use the same conditions, for example, determination methods for a sample obtained from a subject who was administered the compound and a control sample. Likewise, the same antibodies are used in both cases.

The greater degree of binding (increased binding) of the detected antibody to the sample from the patient to whom the compound was administered, compared with the binding of the antibody to the control sample, indicates binding of the compound to the target, a trimeric member of the TNF superfamily. In other words, an equivalent or reduced degree of binding (reduced binding) in a sample from a patient administered a compound compared to a control indicates that the compound does not bind to the target. In other words, if no significant difference is observed between the two degrees of binding, then this indicates the absence of binding to the target.

One skilled in the art can easily determine when increased binding is observed compared to the control. For example, if the control is a range of data, the interaction with the target can be determined based on the spread of the data, the difference between the control data and the detected level of antibody binding in the test sample and the calculated confidence intervals. It is also possible to identify an interaction with a target if the detected level of binding in the test sample is higher than the maximum level of binding detected in any negative control.

Target binding can be detected if the level of antibody binding increases by approximately 30% or more compared to the highest level of binding in the control range. Target binding can also be detected if the level of antibody binding increases by about 40% or more, or by about 50% or more, relative to the control range. The same applies when the control is the average value or one value obtained from the analysis of a sample from a patient that was obtained before administration of the compound. It should be understood that there is no upper limit to the increase in binding (percentage) compared to the control.

Antibodies can be used to screen for a compound that causes a conformational change in a trimeric member of the TNF superfamily, wherein the conformational change modulates signaling through a desired TNF superfamily receptor upon binding of a trimeric member of the TNF superfamily. The superfamily member is typically TNFa and/or the modulation is antagonism of signaling through the TNFR1 receptor.

Antibodies can be used to treat and/or prevent a pathological condition. Accordingly, an antibody is provided for use in a method of treating a human or animal. A method of treatment is also provided which consists of administering an antibody to the subject. The antibody can be used in any therapeutic indication and/or pharmaceutical composition described in this context.

Antibody assays.

As described herein, antibodies are provided that selectively bind to at least one compound-trimer complex as described herein versus binding to only one compound or to a member of the TNF superfamily in the absence of a compound. These antibodies can be used to identify other compounds or classes of compounds that have identical properties.

Monoclonal antibodies against a member of the TNF superfamily can be obtained using standard techniques, as described in this context. These antibodies against a member of the TNF superfamily can then be screened for antibodies that bind to compound-trimer complexes or monoclonal antibodies whose binding to a member of the TNF superfamily is inhibited by compounds as described herein.

Alternatively, monoclonal antibodies can be generated against specific TNF superfamily member trimer-compound complexes. These antibodies can then be screened for monoclonal antibodies that selectively bind to a member of the TNF superfamily in the presence of the compound compared to its binding to a member of the TNF superfamily in the absence of the compound.

When an antibody is produced that selectively binds to at least one compound-trimer complex over binding to the compound alone or to a member of the TNF superfamily in the absence of the compound, such compounds can be used in the screening of other compounds.

- 23 045234 having the same activity as the compounds under study.

Thus, an assay for identifying a compound of the invention is provided which includes the following steps:

a) performing a binding assay to assess the binding affinity of the compound-trimer complex of interest to the antibody,

b) comparing the binding affinity measured in step (a) with the binding affinity of another compound-trimer complex known to bind the antibody of step (a) with high affinity, and

c) selecting the compound present in the compound-trimer complex in step (a) if its measured binding affinity is acceptable given the comparison made in step (b).

It should be understood that the term different compound-trimer complex used above in the description of step (b) generally refers to a complex containing the same trimer as the compound-trimer complex used in step (a) but a different compound. The compound may be any of compounds (1)-(6).

The term acceptable as used in the description of step (c) means that the binding affinity of the compound-trimer complex used in step (a) and the binding affinity of the other compound-trimer complex used in step (b) are substantially comparable.

The selectivity of the binding of the specified antibody to the specified complex is usually measured relative to the binding of the specified antibody to a member of the TNF superfamily in the absence of a compound or a compound in the absence of a member of the TNF superfamily.

The binding affinity of the compound-trimer complex used in step (a) generally exceeds the binding affinity of the other compound-trimer complex used in step (b). Accordingly, the increase in binding affinity of the compound-trimer complex used in step (a) compared to the binding affinity of the other compound-trimer complex used in step (b) is in the range of 10-, 20-, 50-, 100-, 200- or 500x range.

Libraries of compounds can be analyzed using antibodies.

Library compounds can be incubated in the presence of the specified antibody in the presence or absence of a member of the TNF superfamily. Compounds that form part of a compound-trimer complex that binds to an antibody only in the presence of both a member of the TNF superfamily and the compound are candidates that have the same activity as the compounds described herein. The methods described herein can be used to detect a test compound that is a compound described herein.

One or more antibodies may be used in the assay. Antibody assays can use a generic antibody that is capable of binding to complexes of any compound with a specific member of the TNF superfamily.

An antibody assay can use a panel of several antibodies that specifically bind to different compound-trimer complexes. The antibody panel may include at least

5, at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 antibodies (eg, up to 75 antibodies).

An antibody assay can be a high-throughput assay that screens multiple test compounds in a short period of time to identify compounds.

Members of the TNF superfamily and their receptors can be purified or they can be present in mixtures such as cultured cells, tissue samples, body fluids or culture media. Assays can be developed that are qualitative or quantitative assays, the latter being useful for determining the binding parameters (affinity constants and kinetic constants) of the compound of interest to the trimeric forms of members of the TNF superfamily, as well as the binding parameters of the compound-trimer complex to the desired TNF receptor.

A sample containing a member of the TNF superfamily and a compound may further contain a destabilizing agent. Destabilizing agents, also known as chaotropic agents, include agents such as urea, guanidine, or acetonitrile used at low molar concentrations (e.g., 1 M), where the use of these agents at high concentrations (e.g., 6 M or more) results in complete dissociation of the TNFa trimer and unfolding of its constituent monomeric TNFa subunits. DMSO can be used as a destabilizing agent, usually at a concentration of 5, 10% or more.

Test compounds may have any/all of the properties described above.

TNF superfamily and its receptors.

There are currently 22 known members of the TNF superfamily:

- 24 045234

TNF oc (TNFSF1A), TNFp (TNFSF1B), CD40L (TNFSF5), BAFF (TNFSF 13B/BlyS), APRIL (TNFSF 13), OX40L (TNFSF4), RANKL (TNFSF 11/TRANCE), TWEAK (TNFSF 12), TRAIL (TNFSF 10), TL1A (TNFSF15), LIGHT (TNFSF 14), lymphotoxin, lymphotoxin β (TNFSF3), 4-1BBL (TNFSF9), CD27L (TNFSF7), CD30L (TNFSF8), EDA (ectodysplasin), EDA-A1 ( ectodysplasin A1), EDA-A2 (ectodysplasin A2), FASL (TNFSF6), NGF and GITRL (TNFSF 18).

The TNF superfamily member is typically TNFa. TNFa exists in both a soluble form (TNFas) and a membrane-bound form (TNFam). The term TNFa as used in this context includes (both) TNFas and TNFam forms. The most suitable TNFa is the TNFas form. TNFas may contain the sequence SEQ ID NO: 35 or SEQ ID NO: 36, or variants thereof (as described above).

The assays can be used to identify modulators of at least one of any members of the TNF superfamily, including the 22 known members of the TNF superfamily. In more detail, the assays can be used to identify compounds that bind to any member of the TNF superfamily, primarily the trimeric forms of members of the TNF superfamily, as well as compounds that stabilize these trimers in a conformation in which they are capable of binding to the desired TNF receptor, and that modulate signal transmission through the specified receptor. First of all, the assay of the invention is used to identify modulators of TNFa or CD40L, especially TNFa or even TNFa _s .

The compound described herein may be a modulator of at least one of any members of the TNF superfamily, including the 22 known members of the TNF superfamily. In more detail, a member of the TNF superfamily is TNFa or CD40L, especially TNFa or even TNFas.

The compound-trimer complex may include the trimeric form of any member of the TNF superfamily, including the 22 known members of the TNF superfamily. The TNF superfamily member is typically TNFa or CD40L. The TNF superfamily member may be TNFa, most preferably TNFa _s .

Members of the TNF superfamily bind to TNF receptors and initiate signal transduction through them. There are currently 34 known TNF receptors:

41BB (TNFRSF9/CD137), NGF R (TNFRSF16), BAFF R (TNFRSF13C), osteoprotegerin (TNFRSF1 IB), BCMA (TNFRSF 17), 0X40 (TNFRSF4), CD27 (TNFRSF7), RANK (TNFRSF1 1A), CD30 (TNFRSF8 ), RELT (TNFRSF 19L), CD40 (TNFRSF5), TACI (TNFRSF 13B), DcR3 (TNFRSF6B), TNFRH3 (TNFRSF26), DcTRAIL RI (TNFRSF23), DcTRAIL R2 (TNFRSF22), TNF-R1 (TNFRSF 1 A), TNF-R2 (TNFRSF IB), DR3 (TNFRSF25), TRAIL RI (TNFRSF 1 OA), DR6 (TNFRSF21), TRAIL R2 (TNFRSF 1 OB), ED AR, TRAIL R3 (TNFRSF IOC), Fas (TNFRSF6/CD95) , TRAIL R4 (TNFRSF 10D), GITR (TNFRSF 18), TROY (TNFRSF 19), HVEM (TNFRSF 14), TWEAK R (TNFRSF 12A), TRAMP (TNFRSF25), lymphotoxin β R (TNFRSF3), and XEDAR.

A suitable TNF receptor is TNF-R1 (TNFR1) or TNF-R2 (TNFR2). The term TNF-R as used herein includes TNF-R1 and TNF-R2, including the extracellular domain (ECD) of TNF-R1 and TNF-R2. The assays can be used to identify compounds that modulate signaling by members of the TNF superfamily through any desired TNF superfamily receptor. Assays can be used to identify compounds that modulate signaling by members of the TNF superfamily through TNF-R1, TNF-R2, or CD40. The TNF superfamily member may be TNFa, and the TNF receptor may be TNF-R1 or TNF-R2. First, the TNF superfamily member may be TNFa, and the TNF receptor may be TNF-R1. First, the TNF superfamily member may be TNFa _s and the TNF receptor may be TNF-R1. Assays can be used to identify compounds that act by specifically modulating signal transduction by members of the TNF superfamily through TNF-R1. First, the compounds may have effects by modulating signaling by TNF superfamily members via TNF-R1, but not affecting signaling by TNF superfamily members via TNF-R2.

The compound-trimer complex can modulate signaling by members of the TNF superfamily through at least one TNF receptor, including the 34 known TNF receptors. The TNF receptor is usually TNF-R1, TNF-R2 or CD40L.

- 25 045234

First of all, the TNF superfamily member is TNFa, and the TNF receptor is TNF-R1 or TNF-R2. Even more preferably, a member of the TNF superfamily is

TNFa, and the TNF receptor is TNF-R1. The most useful member of the TNF superfamily is TNFas, and the TNF receptor is TNF-R1.

Therapeutic indications.

TNFa is the original member of the TNF superfamily. TNFa is a pleiotropic cytokine that mediates immune regulation and inflammatory responses. It is also known that TNF in vivo is involved in the development of responses to bacterial, parasitic and viral infections. First of all, TNFa is known to be involved in the development of rheumatoid arthritis (RA), inflammatory bowel diseases (including Crohn's disease), psoriasis, Alzheimer's disease (AD), Parkinson's disease (PD), pain, epilepsy, osteoporosis, asthma, sepsis, fever, systemic lupus erythematosus (SLE) and multiple sclerosis (MS), and cancer. TNFa is also known to be involved in the development of amyotrophic lateral sclerosis (ALS), ischemic stroke, immune complex glomerulonephritis, lupus nephritis (LN), glomerulonephritis associated with antibodies against neutrophil cytoplasmic components (ANCA-), lipoid nephrosis, diabetic nephropathy (DN) , acute kidney injury (AKI), obstructive uropathy, renal allograft rejection, cisplatin-induced AKI and obstructive uropathy.

Other members of the TNF superfamily are known to be involved in the development of autoimmune disease and immunodeficiency conditions. First of all, members of the TNF superfamily are known to be involved in the development of RA, SLE, cancer, MS, asthma, rhinitis, osteoporosis and multiple myeloma (MM). TL1A is known to be involved in the development of organ transplant rejection.

The compound described herein can be used to treat, prevent, or ameliorate any condition that can be treated, prevented, or ameliorated by a standard modulator of a member of the TNF superfamily. The compound can be used alone or in combination with a standard TNF superfamily member modulator. Any condition that develops as a result of partial or wholly pathogenic signaling by a member of the TNF superfamily through the TNF receptor, or due to insufficient signaling by a member of the TNF superfamily through the TNF receptor, can in principle be treated, prevented, or reduce the intensity of its symptoms. Pathogenic signaling by a member of the TNF superfamily through the TNF receptor involves increased signaling through the TNF receptor, i.e. exceeding the normal physiological level of signal transduction includes TNF receptor signaling that was initiated normally but which does not stop in response to normal physiological signals, as well as TNF receptor signaling that is within the normal physiological range of magnitude but which has been initiated by non-physiological factors. Preferably, this relates to the treatment, prevention or reduction of symptoms of conditions that are mediated by or influenced by TNFa or CD40L.

Compounds that interact with TNFa will accordingly have beneficial effects in the treatment and/or prevention of various human diseases. These include autoimmune and inflammatory diseases, neurological and neurodegenerative disorders, pain and nociceptive disorders, and cardiovascular disorders.

Inflammatory and autoimmune disorders include systemic autoimmune disorders, autoimmune endocrine disorders, and organ-specific autoimmune disorders. Systemic autoimmune disorders include systemic lupus erythematosus (SLE), psoriasis, vasculitis, polymyositis, scleroderma, multiple sclerosis, ankylosing spondylitis, rheumatoid arthritis, and Sjogren's syndrome. Autoimmune endocrine disorders include thyroiditis. Organ-specific autoimmune disorders include Addison's disease, hemolytic or pernicious anemia, glomerulonephritis (including Goodpasture's syndrome), Graves' disease, idiopathic thrombocytopenic purpura, insulin-dependent diabetes mellitus, juvenile diabetes, uveitis, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), pemphigus, atopic dermatitis, autoimmune hepatitis, primary biliary cirrhosis, autoimmune pneumonia, autoimmune carditis, myasthenia gravis, spontaneous infertility, osteoporosis, asthma, and muscular dystrophy (including Duchenne muscular dystrophy).

Neurological and neurodegenerative disorders include Alzheimer's disease, Parkinson's disease, Huntington's disease, stroke, amyotrophic lateral sclerosis, spinal cord injury, head injury, seizures and epilepsy.

Cardiovascular disorders include thrombosis, cardiac hypertrophy, hypertension, irregular heartbeat (eg, heart failure), and sexual disorders (including erectile dysfunction and female sexual dysfunction).

First of all, the compound can be used for the treatment or prevention of inflammatory diseases, central nervous system disorders, immune disorders and autoimmune diseases, pain, osteoporosis,

- 26 045234 fever and transplant rejection. The compound can be used for the treatment or prevention of rheumatoid arthritis, inflammatory bowel diseases (including Crohn's disease), psoriasis, Alzheimer's disease, Parkinson's disease, epilepsy, asthma, sepsis, systemic lupus erythematosus, multiple sclerosis, asthma, rhinitis, cancer and osteoporosis. The compound can be used for the treatment or prevention of rheumatoid arthritis (RA), nonspecific inflammatory arthritis, erosive bone disease, chondritis, cartilage degeneration and/or degradation, juvenile inflammatory arthritis, Still's disease (in children and/or adults), juvenile idiopathic arthritis, juvenile idiopathic arthritis (both oligoarticular and polyarticular forms), inflammatory bowel diseases (including Crohn's disease, ulcerative colitis, indeterminate colitis, inflammation of the ileoanal pouch), psoriasis, psoriatic arthropathy, ankylosing spondylitis, Sjögren's disease, Alzheimer's disease (AD), Behçet's disease, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), ischemic stroke, pain, epilepsy, osteoporosis, osteopenia, anemia of chronic disease, cachexia, diabetes, dyslipidemia, metabolic syndrome, asthma, chronic obstructive airway disease (or pulmonary disease), sepsis, fever, respiratory distress syndrome, systemic lupus erythematosus (SLE), multiple sclerosis (MS), immune complex glomerulonephritis, lupus nephritis (LN), glomerulonephritis associated with antibodies against neutrophil cytoplasmic components (ANCA-), lipoid nephrosis, diabetic nephropathy (DN), acute kidney injury (AKI), obstructive uropathy, renal allograft rejection, cisplatin-induced AKI and obstructive uropathy, eye diseases (including diabetic retinopathy, diabetic macular edema, retinopathy of prematurity, age-related macular degeneration, macular edema , proliferative and/or non-proliferative retinopathy, corneal vascularization, including neovascularization, retinal vein occlusion, various forms of uveitis and keratitis), thyroiditis, fibrotic disorders, including various forms of liver fibrosis, various forms of pulmonary fibrosis, systemic sclerosis, scleroderma, cancer and complications, associated with cancer (including skeletal complications, cachexia and anemia).

As discussed above, antibodies can be used as biomarkers of target binding to assess the effectiveness of treatment with a compound or complex as described herein. In one embodiment, a sample taken from a subject who has been treated with a compound or complex as described herein may be contacted with the antibody. The antibody can then be used to determine the amount of TNF superfamily member-compound complex present in the sample. The amount of complex determined using the antibody can be used as an indicator of the effectiveness of treatment. For example, the greater the amount of complex detected by antibodies, the higher the effectiveness of treatment. The amount of complex determined using antibodies is directly proportional to the effectiveness of treatment. For example, a twofold increase in the amount of the complex determined using antibodies may indicate a twofold increase in the effectiveness of treatment.

The antibody can be used to determine the amount of compound-trimer complex using any suitable technique. Standard techniques are known in the art and are described herein. For example, ELISA and Western blotting using antibodies of the invention can be used to determine the amount of compound-trimer complex.

The amount of compound-trimer complex can be determined by measuring the mass of the compound-trimer complex, the concentration of the compound-trimer complex, and the molarity of the compound-trimer complex. The indicated quantity can be represented in any appropriate units. For example, the concentration of the compound-trimer complex can be expressed in pg/ml, ng/ml or μg/ml. The mass of the compound-trimer complex can be expressed in pg, ng or μg.

The amount of compound-trimer complex in a test sample can be compared with the level of compound-trimer complex in another sample, such as a control sample, as described herein. This technique can estimate the actual amount of compound-trimer complex, eg, mass, molar amount, concentration, or molarity of compound-trimer complex in samples. The amount of a compound-trimer complex can be compared to that in another sample without quantifying the mass, molar amount, concentration, or molarity of the compound-trimer complex. Thus, the amount of compound-trimer complex in a sample can be estimated in terms of a relative amount, such as relative mass, relative molar amount, relative concentration, or relative molarity of the compound-trimer complex when comparing two or more samples.

Pharmaceutical compositions, dosages and dosage regimens.

The antibody, compound or complex can be processed into a pharmaceutical composition. The pharmaceutical composition is normally sterile and typically includes a pharmaceutically acceptable carrier and/or adjuvant. The pharmaceutical composition may further comprise a pharmaceutically acceptable adjuvant and/or carrier.

The term pharmaceutically acceptable carrier as used in this context includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterials

- 27 045234 anal and antifungal agents, isotonic agents and absorption retarding agents, etc. As the carrier, a carrier suitable for parenteral administration, for example, such as intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or infusion, can be used. Alternatively, the carrier may be a carrier suitable for non-parenteral administration, such as a topical, epidermal or mucosal route. As the carrier, a carrier suitable for oral administration can be used.

Depending on the route of administration, the modulator may be coated with a material to protect the compound from acids and other natural conditions that may inactivate the compound.

Pharmaceutical compositions may include one or more pharmaceutically acceptable salts. The term pharmaceutically acceptable salt means a salt that retains the desired biological activity of the parent compound and does not cause any undesirable toxicological effects. Examples of these salts include acid addition salts and base addition salts.

Pharmaceutically acceptable carriers include aqueous-based carriers or diluents. Examples of suitable aqueous-based carriers that can be used in pharmaceutical compositions include water, aqueous buffer solutions and saline. Examples of other carriers include ethanol, polyols (such as glycerin, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. In many cases, it is desirable to include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride.

Therapeutic compositions generally must be sterile and stable under conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for incorporating the drug at high concentrations.

Pharmaceutical compositions may contain additional active ingredients.

The invention describes kits containing antibodies, compounds and/or complexes of the invention and instructions for use. The kit may further contain one or more additional reagents, such as an additional drug or prophylactic agent, as described above.

Compounds identified by methods and/or antibodies, and antibodies or formulations or compositions thereof can be administered for prophylaxis and/or treatment.

In treatment, the compounds are administered to a subject already suffering from a disorder or condition as described above in an amount sufficient to treat, alleviate or partially arrest the progression of the condition or reduce the intensity of one or more of its symptoms. This treatment may result in a reduction in the severity of symptoms of the disease or an increase in the frequency or duration of symptom-free periods. An amount sufficient to achieve these purposes is defined as a therapeutically effective amount.

In prophylactic treatment, the compositions are administered to a subject at risk of developing a disorder or condition as described above in an amount sufficient to prevent or reduce subsequent complications of the condition or one or more symptoms thereof. An amount sufficient to achieve the stated objectives is defined as a prophylactically effective amount. Effective amounts to achieve each purpose will depend on the severity of the disease or injury, as well as the body weight and general condition of the subject.

The subject to which the drug is administered is a human or non-human animal. The term non-human animal includes all vertebrates, such as mammals and non-mammals such as non-human primates (lower primates), sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. .d. Typically, the subject to whom administration is performed is a human.

The compound or pharmaceutical composition can be administered by one or more routes of administration using one or more of a variety of methods known in the art. It will be apparent to one skilled in the art that the route and/or method of administration can be varied depending on the desired results.

Examples of routes of administration of compounds or pharmaceutical compositions include intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or infusion. The term parenteral administration, as used in this context, refers to routes of administration that are distinct from enteral and topical administration, and is typically accomplished by injection. In another embodiment, the compound or pharmaceutical composition can be administered by a non-parenteral route, such as a topical, epidermal or mucosal route. The compound or pharmaceutical composition can also be used for oral administration.

The practitioner can determine the appropriate dose of the compound or pharmaceutical component

- 28 045234 positions. The actual dosage levels of the active ingredients in the pharmaceutical compositions can be varied to provide compositions containing the active ingredient in an amount effective to achieve the desired therapeutic response for a particular patient without the composition and route of administration being toxic to the patient. The dosage level selected depends on a variety of pharmacokinetic factors, including the potency of the specific compositions, route of administration, time of administration, elimination rate of the particular compound used, duration of treatment, other drugs, compounds and/or materials used in combination with the particular combinations used, age, gender , body weight, condition, general condition and medical history of the patient requiring such treatment, and other similar factors well known in medicine.

A suitable dose may be selected, for example, from about 0.01 μg/kg to about 1000 mg/kg body weight, typically from about 0.1 μg/kg to about 1000 mg/kg body weight of the patient requiring such treatment. For example, a suitable dose may be from about 1 μg/kg to about 10 mg/kg body weight per day, or from about 10 μg/kg to about 5 mg/kg body weight per day.

Dosage regimens may be adjusted to provide the optimal desired response (eg, therapeutic response). For example, a single dose may be administered, multiple divided doses may be administered over a period of time, or the dose may be proportionally decreased or increased depending on the therapeutic situation. The term unit dosage form as used herein refers to physically discrete forms suitable for use as unitary doses administered to subjects in need of such treatment, each form containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in the mixture with the necessary pharmaceutical carrier.

The drug may be administered as a single dose or multiple doses. Multiple doses may be administered by the same or different routes and to the same or different areas of the body. Alternatively, dosages may be provided as a sustained release formulation, in which case less frequent administration is required. The dosage and frequency of administration may be adjusted depending on the patient's half-life of the antagonist and the desired duration of treatment.

As described above, the compounds or pharmaceutical composition can be administered in conjunction with one or more other therapeutic agents. For example, the other agent may be an analgesic, anesthetic, immunosuppressant, or anti-inflammatory agent.

The combined administration of two or more agents can be accomplished in a number of different ways. Both agents may be administered together in a single composition, or they may be administered in separate compositions in combination therapy. For example, one agent may be administered before, after, or concurrently with another agent.

The following examples are presented to illustrate the present invention only.

Examples

Example 1.

Synthesis of compounds.

The synthesis of compound (1) is described in WO 2013/186229 (example 44).

The synthesis of compound (2) is described in WO 2013/186229 (Example 89).

The synthesis of compound (3) is described in WO 2014/009295 (example 129).

The synthesis of compound (4) is described in WO 2014/009295 (example 173).

The synthesis of compound (5) is described in WO 2014/009295 (example 319). The synthesis of compound (6) is described in WO 2013/186229 (example 490).

Example 2.

Obtaining antibodies.

After immunization of 5 Sprague Dawley rats with a complex of human TNFa with a compound, benzimidazole (1), immune B cells were cultured in 96-well plates to induce cloning and antibody secretion (see Tickle S. et al., High throughput screening for high affinity antibodies, Journal of Laboratory Automation, 14, 303-307 (2009)). The culture supernatants were then screened for the presence of IgG antibodies that preferentially bind to human TNFa complexed with compound (1) (50-fold molar excess) compared to human TNFa using a homogeneous particle FMAT assay (Fluorescent Microvolumetric Assay Technology). Human TNFa (± compound (1)) was adsorbed onto the surface of particles (Bangs superavidin-coated Bangs Beads, catalog number CP01N) using a coupling system and a human TNF-receptor I-Fc fusion protein (R&F D Systems, catalog number 372-R1-050), coupled with biotinylated anti-human Fc antibodies (Jackson, catalog number 109-066-098).

Antibodies that were characterized by preferential binding to the TNFa-29 045234 compound (1) complex were called conformation-selective and were used for further cloning. The foci fluorescence method (see US Pat. No. 7,993,864/EP157O267B1) was used to identify and isolate antigen-specific B cells from positive wells, and specific antibody variable region genes were isolated from individual cells using reverse transcription (RT) PCR.

The amino acid sequences of two representative antibodies, CA185_01974 and CA185_01979, which are characterized by conformation-selective binding to human and mouse TNFa + compound, are presented below:

CA185_01974.0 (VR0001837).

Light Chain Variable Region (LCVR), SEQ ID NO: 7 (CDRs are underlined)

DIOMTOSPASLPASPEEIVTITCQASQDIGNWLSWYOOKPGKSPOLLIYGATSL

ADGVPSRFSASRSGTOYSLKISRLOVEDFGIFYCLQGOSTPYTFGAGTKLELK

Heavy Chain Variable Region (HCVR), SEQ ID NO: 8 (CDRs are underlined) DVQLVESGGGLVQPGRSLKLSCAASGTFFSAYYMAWVRQAPTKGLEWVASI

NYDGANTFYRDSVKGRFTVSRDNARSSLYLQMDSLRSEDTATYYCTTEA

YGYNSNWFGYWGQGTLVTVSS

CA185_01979.0 (VR0001842)

Light Chain Variable Region (LCVR), SEQ ID NO: 22 (CDRs are underlined)

DIOMTQSPASLSASLEEIVTITCQASQDIGNWLSWYOQKPGKSPHLLIYGTTSL

ADGVPSRFSGSRSGTQYSLKISGLQVADIGIYVCLQAYSTPFTFGSGTKLEIK

Heavy Chain Variable Region (HCVR), SEQ ID NO: 23 (CDRs are underlined) EVHLVESGPGLVKPSQSLSLTCSVTGYSITNSYWDWIRKFPGNKMEWMGYIN

YSGSTGYNPSLKSRISISRDTSNNQFFLQLNSITTEDTATYYCARGTYGYNAYH

FD YWGRGVMVT VS S

Example 3.

Putative epitope of the obtained antibodies.

Given the ability of rat-derived antibodies to bind to both human and mouse TNFa in the presence of compounds, detailed analysis of the amino acid sequences of rat, mouse and human TNFa, as well as X-ray diffraction analysis of these TNFa, was performed to identify the putative epitope.

Source - rat, UniProt P16599 (SEQ ID NO: 32)

20 30 40 5060

MSTESMIRDV ELAEEALPKK MGGLQNSRRC LCLSLFSFLL VAGATTLFCL LNFGVIGPNK

80 90 100 110120

EEKFPNGLPL ISSMAQTLTL RSSSQNSSDK PVAHWANHQ AEEQLEWLSQ RANALLANGM

130 140 150 160 170180

DLKDNQLWP ADGLYLIYSQ VLFKGQGCPD YVLLTHTVSR FAISYQEKVS LLSAIKSPCP

190 200 210 220230

KDTPEGAELK PWYEPMYLGG VFQLEKGDLL SAEVNLPKYL DITESGQVYFGVIAL

Source - mouse, UniProt P06804 (SEQ ID NO:33)

20 30 40 5060

MSTESMIRDV ELAEEALPQK MGGFQNSRRC LCLSLFSFLL VAGATTLFCL LNFGVIGPQR

80 90 100 110120

DEKFPNGLPL ISSMAQTLTL RSSSQNSSDK PVAHWANHQ VEEQLEWLSQ RANALLANGM

130 140 150 160 170180

DLKDNQLWP ADGLYLVYSQ VLFKGQGCPD YVLLTHTVSR FAISYQEKVN LLSAVKSPCP

190 200 210 220230

KDTPEGAELK PWYEPIYLGG VFQLEKGDQL SAEVNLPKYL DFAESGQVYF GVIAL

Source - human, UniProt P01375 (SEQ ID NO: 34)

- 30 045234

20 30 40 5060

MSTESMIRDV ELAEEALPKK TGGPQGSRRC LFLSLFSFLI VAGATTLFCL LHFGVIGPQR

80 90 100 110120

EEFPRDLSLI SPLAQAVRSS SRTPSDKPVA HVVANPQAEG QLQWLNRRAN ALLANGVELR

130 140 150 160 170180

DNQLVVPSEG LYLIYSQVLF KGQGCPSTHV LLTHTISRIA VSYQTKVNLL SAIKSPCQRE

190 200 210 220230

TPEGAEAKPW YEPIYLGGVF QLEKGDRLSA EINRPDYLDF AESGQVYFGIIAL

By aligning and comparing rat, mouse and human TNFa sequences from the UniProt database, examples were identified where the amino acid sequence of the rat protein differs from the human protein sequence, and where the human and mouse protein sequences are identical in the mature digest product, which include N168, 1194 , F220 and A221 (residues and numbering from the human protein sequence).

These residues were noted in the crystal structure of human TNFa (1TNF) (see Fig. 1). It is possible that any of the isolated amino acid residues is part of the epitope to which antibodies CA185_01974 and CA185_01979 bind.

After cloning the variable regions of the antibodies into mouse IgG and Fab (no hinge region) vectors, the conformation-selective binding nature of antibodies CA185_01974 and CA185_01979 was confirmed using a variety of TNFa-related compounds of interest using HPLC, BIAcore, ELISA and cell-based assays.

Example 4.

High performance liquid chromatography (HPLC) for antibody characterization

Specific binding of mouse Fab fragments was determined by the formation of complexes of the antibody CA185_01974 and the complex of human TNFa and compound (1) using size exclusion chromatography. The results are presented in Fig. 2. According to the data presented in the above figure, when using Fab fragments in 0.5-fold molar excess, the predominant peak in the chromatogram corresponds to the Fab fragment associated with the compound-trimer complex (although there is a small peak, which indicates the presence of some compound complex -trimer that did not bind to the Fab fragment). When using Fab fragments in a 1.0-fold molar excess, one high-molecular-weight peak is observed in the chromatogram, corresponding to the Fab fragment associated with the trimer-compound complex. When using Fab fragments in 1.5- and 2-fold molar excess, a low molecular weight peak is observed in the chromatogram, corresponding to an unbound Fab fragment.

Thus, at 1.5- and 2-fold molar excess of Fab fragments, the stoichiometric ratio of Fab: TNFa trimer is 1:1.

The binding of CA185_01979 to the complex of human TNFa and compound (1) was also studied using size exclusion chromatography. The results are presented in Fig. 3. In the case of using CA185_01974 with a 1.5- and 2-fold molar excess of Fab fragments, the stoichiometric ratio of Fab: TNFa trimer is 1:1.

Example 5.

BIAcore assays for antibody characterization.

Surface plasmon resonance analysis was performed at 25°C using a BIAcore T200 (GE Healthcare). Anti-mouse Fc antibodies (Jackson, 115-006-071) were immobilized onto the CM5 sensor chip (GE Healthcare) using the amine condensation method, with an immobilization level of 6000 response units. HBS-EP buffer solution (10 mM HEPES (pH 7.4), 0.15 M NaCl, 3 mM EDTA, 0.05% (v/v) detergent P20 (GE Healthcare) + 1% DMSO was used as working buffer solution.10 μl of each IgG (1 μg/ml) was pumped into the installation, and they were bound to immobilized anti-mouse Fc antibodies, thereby obtaining a TNFa-binding surface.

Human or mouse TNFa (laboratory prepared, 50 nM) was preincubated in HBS-EP+ buffer solution (1% DMSO) containing compound (2 μM) for 5 h.

A solution of human or mouse TNFa ± test compound was passed through each immobilized IgG for 3 min at a flow rate of 30 μl/min. To regenerate the surface, a solution of 40 mM HClx2 and a solution of 5 mM NaOH were pumped through the cell for 60 s for 60 s at a flow rate of 10 μL/min. Double background subtraction curves were analyzed using T200 Evaluation software (version 1.0). Kinetic parameters were determined using a selection algorithm.

Data on the binding kinetics of human and mouse TNFa in the presence and absence of the studied compounds from two series of chemical compounds are presented below in table. 1 and 2.

Table 1

Data from human TNFa analysis using BIAcore

Antibody Human TNFa ka (M^s' ¹ ) kd (with KD (M) CA185_01974 + connection(2) 4.2χ10 ⁵ 3.9χ10' ⁵ 9.4x10’“ CA185_01974 + connection(1) 3.2x10 ⁵ 3.8x10' ⁵ 1.2x10' ¹⁰ CA185_01974 apo 6.6x10 ⁵ .4 1.3x10 1.9χ10' ⁸ CA185_01979 + connection(2) 5.7x10 ⁵ 3,ZxYu' ⁵ 5.8x10'“ CA185_01979 + connection(1) 4.7χ10 ⁵ 1.6χ 10' ⁵ 3.4x10'“ CA185_01979 apo 1.1X TO ⁵ 7.1x10' ⁴ 6.7x10' ⁹

Both antibodies, CA185_01974 and CA185_01979, are characterized by selective binding (>2 log) to human TNFa, with an altered conformation after binding to typical test compounds from the two chemical series.

table 2

Mouse TNFa analysis data using the BIAcore installation

Antibody Human TNFa ka (M^s' ¹ ) kd (c' ¹ ) KD (M) CA185_01974 + connection (2) 4.2χ10 ⁵ 3.9χ10' ⁵ 9.4x10'“ CA185_01974 + connection (1) 3.2χ10 ⁵ 3.8χ10' ⁵ 1.2x10' ¹⁰ CA185_01974 apo 6.6x10 ⁵ -3 1.3x10 1.9x10' ⁸ CA185_01979 + connection (2) 5.7x10 ⁵ 3.3χ10' ⁵ 5.8x10'“ CA185_01979 + connection (1) 4.7χ10 ⁵ 1.6x10' ⁵ 3.4x10'“ CA185_01979 apo 1.1 χ 10 ⁵ 7.1x10' ⁴ 6.7x10' ⁹

Both antibodies, CA185_01974 and CA185_01979, exhibit selective binding (>1.5 and >2 log, respectively) to human TNFa, with an altered conformation upon binding to typical test compounds from the two chemical series.

Example 6.

Solid-phase ELISA for determining antibody characteristics.

A sandwich ELISA assay was developed to measure the concentration of TNFa bound to the compounds of the invention using the antibody CA185_01974.0, which specifically recognizes TNFa by its conformation altered after complexation with the compounds. Brief description of the analysis. The surface of the microtiter plate was coated with CA185_01974.0 antibodies to bind TNFa as part of a complex with the compound under study. TNFa was incubated in the presence of the test compound in a 50-fold molar excess overnight at 28°C. After this overnight incubation, serial dilutions of TNFa were prepared in purified human plasma depleted of endogenous TNFa in the presence of heterophile antibody blockers and added to the wells of a plate coated as indicated. TNFa concentration curves were then obtained ranging from 0.78 pg/ml to 50 pg/ml. To detect bound TNFa, we used conjugates of biotinylated polyclonal antibodies against TNFa with streptavidin peroxidase and added the substrate TMB, followed by measurement of the colorimetric signal. The sensitivity of the assay was increased by using tyramide signal amplification and the Perkin Elmer ELAST kit as an additional step between the streptavidin peroxidase and substrate treatment steps.

At the same time, we also developed a solid-phase ELISA analysis method for determining the total content of TNFa (TNFa in free form + TNFa as part of a complex with the test compound). In this assay, the antibodies used to obtain the coating were replaced with commercial anti-TNFa polyclonal antibodies (Invitrogen, AHC3812).

In addition, the sample incubation time was increased to 3 hours. All other steps were performed as described for conformation-specific analysis. This analysis makes it possible to calculate the amount

TNFa in the complex with the test compound as a percentage of the total amount of TNFa.

The results of determining the total TNFa content by ELISA for compounds (3), (4) and (5) are presented in Fig. 4.

The results of conformation-specific ELISA analysis of TNFa in the complexes CA185_01974.0 and compounds (3), (4) and (5) are presented in Fig. 5. The absence of a signal for apo TNFa in this assay indicates specific binding of the antibody CA185_01974 to TNFa, which is associated with the compound. This antibody is capable of recognizing TNFa associated with various test compounds from different chemical series.

Example 7.

Cell-based assays to characterize antibodies.

The ability of recombinant antibodies to bind to TNFa with a changed conformation after binding to the compound was also studied by FACS analysis using human embryonic kidney cells (Jumpln, HEK), which, after induction in the presence of doxycycline at a concentration of 1 μg/ml for 2.5 hours, are characterized by overexpression TNF-RI. HEK cells were trypsinized and incubated for 2 h in a medium that allowed restoration of TNFRI levels that decreased during trypsinization. Human TNFa (2 μg/ml) was preincubated with compound (1) (40 μM) or 0.4% DMSO for 1 h at 37°C. The preincubated mixture was added to the cells for 1 h in an ice bath (1:4 dilution, final concentrations: human TNFa - 0.5 μg/ml ± compound (1) (10 μM) or 0.1% DMSO). Cells were washed, fixed (1.5% PFA (paraformaldehyde)) and stained with antibodies (1 μg/ml or 10 μg/ml) for 1 h in an ice bath. (Secondary antibodies: anti-mouse-Alexa488), before assaying for TNFa receptor bound.

FACS (Fluorescence Activated Cell Sorting) cell staining histograms using CA185_01974 and CA185_01979 (1 μg/ml and 10 μg/ml) shown in FIG. 6 indicate that the antibodies only recognize TNFa that has been preincubated with compound (1). No coloring was observed when DMSO was used as a control.

In addition, it was found that Fab fragments CA185_01974 and CA185_01979 specifically bind to membrane-bound TNFa, with a conformation changed due to binding to the compound. Genetically engineered cells with a knockout of the TACE cleavage site of the NS0 cell line, which is characterized by overexpression of membrane TNFa, were incubated with compound (1) (0.001 - 10 μM) or 0.1% > DMSO for 1 hour at 37°C. Cells were washed, fixed, and stained with Fab antibody fragments (0.01 μg/ml or 0.1 μg/ml) for 1 h in an ice bath. (Secondary antibodies: anti-mouse-Fab-DyeLight488 from Jackson ImmunoResearch).

Histograms of FACS staining of cells using Fab fragments CA185_01974 (see Fig. 7) and CA185_01979 indicate that the antibodies only recognize TNF, which was preincubated with compound (1). No coloring was observed when DMSO was used as a control.

Example 8.

Antibodies CA185_01974 show a 300-fold increase in selectivity for the human TNF-compound complex (4)

Compound (4) was incubated with human and cynomolgus TNF (supo) and titrated in the presence of full-length mouse antibody CA185_01974 to determine the exact affinity value. The following controls were used in the experiment: (i) human TNF or cyno + DMSO in the presence of 1974, (ii) human TNF or cyno + DMSO without antibodies, and (iii) human TNF compound or cyno + compound (4) without antibodies. The analysis of each sample and control was carried out in duplicate and in each case four concentrations were tested.

According to the data presented in Fig. 8 and 9, during the assay, background binding of hTNF + compound (4) and cTNF + compound (4) increased by 5-10 RU (resonance units). This effect is evidenced by a higher signal level upon binding of the h/cTNF + compound (4) complex to CA185_01974 in the second repeat. When hTNF and cTNF were bound in the absence of compound (4), a very low level of signal was observed.

According to the above analysis, the binding kinetics of hTNF + DMSO with full-length mouse IgG, CA185_1974_P8, showed similar results consistent with the results described above obtained for samples with one concentration. Regarding the binding affinity of TNF cyno to full-length mouse IgG, CA185_1974_P8, similar results were obtained, but the kinetic parameters were different.

In table Figure 3 shows the parameters of the binding kinetics of each test sample with CA185_1974_P8. In table Figure 4 shows the average values and fold ratio of the parameters of the binding kinetics of various TNF complexes ± compound (4) with CA185_1974_p8. In fig. Figure 8 shows sensorgrams for two repeats of cTNF ± compound (4). In fig. Figure 9 shows sensorgrams for two repetitions of hTNF ± compound (4).

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Table 3 presents the parameters of the binding kinetics of hTNF and cTNF ± compound (4) with antibodies CA185_1974_P8.

Table 3

Repeat Antibody Test sample ka (1/Ms) kd (1/s) KD (M) KD (pm) 1 CA185 1974 P8 cyno TNF 1.03E+05 1.87E-03 1.83E-08 18270 2 CA185 1974 P8 cyno TNF 1.25E+05 1.92E-03 1.54E-08 15350 1 CA185 1974 P8 cyno TNF + connection (4) 1.84E+05 1.46E-05 7.91E-11 79.1 2 CA185 1974 P8 cyno TNF + connection (4) 2.01E+05 2.06E-03 1, OZE-10 103 1 CA185 1974 P8 Human TNF 8.02E+04 1.77E-03 2.21E-08 22100 2 CA185 1974 P8 Human TNF 1.05E+05 1.67E-03 1.59E-08 15900 1 CA185 1974 P8 Human TNF + compound (4) 3.06E+05 1.00E-05 3.27E-11 32.7 2 CA185 1974 P8 Human TNF + compound (4) 3.07E+05 2.73E-05 8.88E-11 88.8

Table 4 presents the average values and fold ratio of the parameters of the binding kinetics of hTNF and cTNF ± compound (4) with CA185_1974_p8.

Table 4

Average of two replicates ka (1/Ms) kd (1/s) KD (M) KD (pm) cyno TNF 1.14E+05 1.90E-03 1.68E-08 16810 cyno TNF + connection (4) 1.93E+05 1.76E-05 9.09E-11 90.92 Multiplicity 1.69 107.81 184.89 184.89 Human TNF 9.27E+04 1.72E-03 1.90E-08 19000 Human TNF + compound (4) 3.06E+05 1.86E-05 6.08E-11 60.76 Multiplicity 3.31 92.43 312.70 312.70

Conclusions.

It was found that antibodies CA185_01974 and CA185_01989 specifically bind to TNFa, with a conformation changed after binding to the compound, and they can be used as biomarkers of target binding for the compounds of the invention.

It has also been established that antibodies bind to TNFa, with a conformation specifically stabilized after binding to compounds from different chemical series. It is envisaged that these antibodies can be used as standards in the determination of such conformations and similar biological conformations of the TNFa trimer stabilized by compounds from a wider range of chemical series than described in this context. Based on the data presented, the human TNFa trimer can be considered to be stabilized in the defined biological conformation described if the binding of antibodies CA185_01974 or CA185_01989 is characterized by a KD value of less than 1 nM, according to the BIAcore analysis described above.

Example 9.

The compounds and complexes described in Ma et al. (2014) and Silvian et al. (2014) have different characteristics compared to the compounds of the present invention

According to the data presented on page 12458 in Ma et al. (JBC, 289, 12457-12466 (2014)), compound C87 was identified during a virtual screen, the purpose of which was to find molecules whose size corresponds to the space occupied by a 7-membered peptide from loop 2/domain 2 of TNFR1, upon its interaction with the outer surface of TNFe. Compound C87, described in Ma et al., and compound BI08898, described in Silvian et al. (ACS Chemical Biology, 6, 636647 (2011)), were studied by the present inventors.

A summary of the results (deviations) obtained.

The results of the Biacore assay for C87 described in the article by Ma et al. could not be repeated. No data on specific inhibition of TNF in cells could be obtained.

In addition, according to mass spectrometry, which has sensitivity for binding affinity at the millimolar level, no binding of C87 was observed.

Extended crystallographic studies were performed only on apo-TNF (TNF in the absence of a compound).

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According to the polarization fluorescence (PF) analysis, no significant inhibition was observed for compound C87, exceeding the level of interference of the compound when reading the fluorescent signal.

According to thermal fluorescence analysis, which allows us to evaluate the stabilization of the melting point of TNFa, a slight stabilization is observed for C87.

Thus, no evidence was found that compound C87 binds at the center of the trimer.

The vast majority of data did not indicate a direct interaction with TNFa. BI08898 was also found not to bind to TNFa.

NFKB reporter gene assay of TNF-induced HEK cells

Before addition to HEK-293 cells stably transfected with SEAP under the control of NFkB, compound C87 was preincubated with TNFa for 1 hour. An appropriate reverse screen was also performed to determine off-target activity that is not associated with TNF. Before assessing the level of inhibition compared to 100% blocking with a control compound, the test sample was incubated overnight. The maximum concentration of C87 was 10,000 nM, and the assay was performed using 3-fold serial dilutions.

No inhibitory effect that was not attributable to off-target activity was observed.

Biacore analysis.

TNF was immobilized using the avi-tag linker and the C87 compound was passed through the chip. In one experiment, the dose-response effect was assessed using C87 at the maximum concentration, which was 10 μM. No binding could be detected.

In the second experiment, the flow rate of the C87 solution passing through the chip was reduced. A slight signal shift was observed, but overall binding remained at a negligible level.

The binding of C87 to TNF described in the paper by Ma et al. is likely to be super-stoichiometric, according to the level of the resonance signal (RU) along the Y axis. At standard TNF densities on the chip, the magnitude of the indicated signal was in the range of values that were thirty times higher than values expected from simple 1:1 binding.

In another experiment, the interaction of BI08898 with the immobilized soluble form of CD40L and the soluble form of TNFa was studied using the SPR (surface plasmon resonance) method on a Biacore 4000 installation. When binding to CD40L, the geometric mean IC50 was 17 μM, while according to the indicated analysis, binding to TNFa in concentrations up to up to 100 µM was not observed.

Mass spectrometry.

Data on the binding of C87 (400 μM) to human TNFa (20 μM) could not be obtained. It turned out that the binding of compounds with low molecular weight (~473 Da) is less than 5% of the receptor occupancy. The molecular weight of C87 is 503 Da. Based on occupancy at a concentration of 400 µM, one would expect the affinity to appear in excess for low molecular weight compounds, i.e. more than 1 mm.

Crystallography.

In general, extensive crystallization studies of C87 with TNFa were conducted, including study conditions typically used with the compounds described in the present invention. These studies consisted of a large number of crystallization experiments using ligands at different concentrations, protein at different concentrations, and at different incubation times. Several crystals were examined and found to be the salt or TNF without the compound.

Polarizing fluorescence (PF)

Before analysis using a fluorescent compound (probe), C87 was preincubated with TNFa for 1 hour. Competition with the fluorescent compound for direct (binding to the same site) or indirect (disruption of TNF) binding was assessed by reducing the level of PF.

By extrapolation of the inhibition curve, the IC ₅₀ was found to be approximately 100 μM. However, fluorescence quenching was observed at the highest inhibitor concentrations, so this subtraction analysis determined that C87 had a negligible inhibitory effect.

Thermal fluorescence analysis.

Thermal fluorescence analysis allows us to evaluate the change in the melting temperature ( _Tm ) of TNFa as a result of interaction with a compound that stabilizes or changes the conformation of the protein. The stabilizing effect when using C87 at a concentration of 500 μM was 3.8 °C, indicating the possibility of weak binding, which may be nonspecific.

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Claims (16)

CLAIM 1. An antibody that selectively binds to a complex comprising (i) trimeric TNFa, and (ii) a compound selected from the group consisting of compounds (1)-(5) or salts, or solvates thereof: - 36 045234 where the antibody includes a combination of sequences HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 SEQ ID NOs: 4/5/6/1/2/3. 2. The antibody according to claim 1, where the antibody contains a pair of sequences HCVR and LCVR SEQ ID NO: 8 and 7. 3. The antibody of claim 1, wherein the antibody comprises a heavy chain of SEQ ID NO: 12 or 13 and a light chain of SEQ ID NO: 11. 4. An antibody that selectively binds to a complex containing (i) trimeric TNFa, and (ii) a compound selected from the group consisting of compounds (1)-(2) or salts, or solvates thereof: or where the antibody contains a combination of sequences HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 SEQ ID NO: 19/20/21/1/17/18. 5. The antibody according to claim 4, where the antibody contains a pair of sequences HCVR and LCVR SEQ ID NO: 23 and 22. 6. The antibody of claim 4, wherein the antibody comprises a heavy chain of SEQ ID NO: 27 or 28 and a light chain of SEQ ID NO: 26. 7. An antibody according to any one of the preceding claims, wherein the antibody selectively binds to the trimer-compound complex over binding to the compound in the absence of the TNFa trimer and/or to the compound in the absence of the TNFa trimer. 8. The antibody of claim 7, wherein the KD-ab value upon binding of the antibody to the trimer-compound complex is reduced by at least 100-fold, or at least 200-fold, compared to the KD-ab value - 37 045234 for binding to a TNFa trimer in the absence of a compound and/or for binding to a compound in the absence of TNFa. 9. An antibody according to any of the preceding paragraphs, which is: (a) a humanized antibody; or (b) a Fab fragment, modified Fab, Fab', modified Fab', F(ab') ₂ , Fv, single domain antibody, or scFv. 10. An isolated polynucleotide encoding an antibody according to any one of claims 1-3. 11. An isolated polynucleotide encoding an antibody according to any one of claims 4-6. 12. Use of an antibody according to any one of claims 1 to 9 as a biomarker of target binding to detect a compound-trimer complex in a sample obtained from a subject, wherein said antibody is detectable, and wherein said complex includes trimeric TNFa and a compound that is capable of binding with trimeric TNFa, and thereby the compound-trimer complex binds to the desired TNF receptor and modulates trimer-induced signal transduction through the receptor. 13. A method for detecting the interaction of a target with a TNFa trimer compound, whereby the compound-trimer complex binds to the cTNF receptor and modulates trimer-induced signal transduction through the receptor, the method comprising: (a) obtaining a sample from a subject who has been administered said compound, (b) contacting the antibody of any one of claims 1 to 9 with said sample and a control sample, wherein said antibody is detectable, (c) determining the level of binding of said detectable antibody with said sample and said control sample, wherein the binding of said detectable antibody to said sample is greater than the binding of said detectable antibody to said control sample, indicating interactions of the target with said compound upon binding to said TNFa trimer. 14. Use of an antibody according to any one of claims 1 to 9 to detect a compound that causes a conformational change in the TNFa trimer, wherein said conformational change modulates signaling through the TNF receptor upon binding to the TNFa trimer. 15. A method for identifying a compound capable of binding to TNFa and modulating TNFa trimer signaling through a receptor, said method comprising the following steps: (a) performing a binding assay to measure the binding activity of the test compound-TNFa trimer complex and the test compound against the antibody of any one of claims 1 to 9, (b) comparing the binding activity measured in step (a) with the binding activity of various compound-trimer complexes known to bind with high activity to the antibody specified in step (a), and (c) selecting the compound present in the compound-trimer complex in step (a) if the measured binding activity is acceptable according to the comparison data specified in stage (b). 16. The method according to claim 15, where the method is a high-speed analysis method. -