mdx‐1097 induces antibody‐dependent cellular cytotoxicity ... · mdx-1097 induces...

MDX-1097 induces antibody-dependent cellular cytotoxicityagainst kappa multiple myeloma cells and its activity isaugmented by lenalidomide

Parisa Asvadi,1 Andrew Cuddihy,2,3

Rosanne D. Dunn,1 Vivien Jiang,1 Mae

X. Wong,1,3 Darren R. Jones,1 Tiffany

Khong2,3 and Andrew Spencer2,3

1Immune System Therapeutics, Sydney, NSW,2Malignant Haematology & Stem Cell Trans-

plantation Service, Alfred Hospital, and 3Mye-

loma Research Group, Australian Centre for

Blood Diseases, Monash University, Melbourne,

VIC, Australia

Received 15 September 2014; accepted for

publication 3 December 2014

Correspondence: Parisa Asvadi, Immune

System Therapeutics (IST) Ltd, Suite 145

National Innovation Centre, Australian

Technology Park, 4 Cornwallis St., Eveleigh,

NSW 2015, Australia.

E-mail: [email protected]; pasvadi@gmail.

com

and

Andrew Spencer, Myeloma Research Group,

South Block, Alfred Hospital, 55 Commercial

Rd., Melbourne, VIC 3004, Australia.

E-mail: [email protected]

Summary

MDX-1097 is an antibody specific for a unique B cell antigen called kappa

myeloma antigen (KMA) that consists of cell membrane-associated free

kappa light chain (jFLC). KMA was detected on kappa human multiple

myeloma cell lines (jHMCLs), on plasma cells (PCs) from kappa multiple

myeloma (jMM) patients and on jPC dyscrasia tissue cryosections. In pri-

mary jMM samples, KMA was present on CD38+ cells that were CD138

and CD45 positive and/or negative. MDX-1097 exhibited a higher affinity

for KMA compared to jFLC and the latter did not abrogate binding to

KMA. MDX-1097-mediated antibody-dependent cellular cytotoxicity

(ADCC) and in vitro exposure of target cells to the immunomodulatory

drug lenalidomide resulted in increased KMA expression and ADCC. Also,

in vitro exposure of peripheral blood mononuclear cells (PBMCs) to lena-

lidomide enhanced MDX-1097-mediated ADCC. PBMCs obtained from

myeloma patients after lenalidomide therapy elicited significantly higher

levels of MDX-1097-mediated ADCC than cells obtained prior to lenalido-

mide treatment. These data establish KMA as a relevant cell surface antigen

on MM cells that can be targeted by MDX-1097. The ADCC-inducing

capacity of MDX-1097 and its potentiation by lenalidomide provide a pow-

erful rationale for clinical evaluation of MDX-1097 alone and in combina-

tion with lenalidomide.

Keywords: antibody therapy, immunotherapy, multiple myeloma.

Multiple myeloma (MM) is an incurable malignancy of ter-

minally differentiated plasma cells (PCs) characterized by PC

accumulation in the bone marrow (BM), lytic bone disease,

renal insufficiency, anaemia, hypercalcaemia and immunode-

ficiency (Katzel et al, 2007; Palumbo & Anderson, 2011). The

BM microenvironment, characterized by the presence of

extracellular matrix proteins and accessory cells, such as BM

stromal cells, osteoclasts and osteoblasts, is believed to play

a significant role in the survival and proliferation of MM

cells (Balakumaran et al, 2010; Palumbo & Anderson, 2011).

In addition to the secretion of an array of cytokines that

stimulate cells within the microenvironment, MM cells

secrete monoclonal immunoglobulin (Ig) (M protein) and/or

Ig light chains which constitute the laboratory hallmark of

MM. The M protein is predominantly of the IgG isotype

while the light chain isotype is either kappa (j) or lambda

(k).

Until the 1990s, MM treatment was restricted to conven-

tional chemotherapy, at which point high-dose therapy with

autologous stem cell transplant (ASCT) and the use of bis-

phosphonates for treatment of MM-related osteolysis were

integrated as standards of care (Kyle & Rajkumar, 2009). In

the past decade, novel agents such as thalidomide, bortezo-

mib and lenalidomide have been introduced into the clinic

with significant benefit in terms of response rates and sur-

vival (Kyle & Rajkumar, 2009). Furthermore, the benefits of

combining various anti-MM agents in comparison to

sequential single agent therapy (Alexania et al, 1977; Lonial

& Kaufman, 2012), and an increased understanding of the

mode of action of novel anti-MM agents (Rajkumar et al,

2005; Quach et al, 2010; Mujtaba & Dou, 2011) has facili-

tated the use of rational approaches in the design of clinical

trials. The use of oncogenomics for identification of high risk

disease (Avet-Loiseau et al, 2007; Shaughnessy et al, 2007)

research paper

ª 2015 John Wiley & Sons Ltd, British Journal of Haematology doi: 10.1111/bjh.13298

and flow cytometry for the detection of minimal residual dis-

ease (Bataille et al, 2006; Rawstron et al, 2008; Paiva et al,

2009) have enabled a more stringent interrogation of new

treatments and their true impact on patient outcome. Coin-

cidentally with the above, monoclonal antibodies have

become an increasingly important class of anti-cancer ther-

apy due to their demonstrable efficacy and favourable toxic-

ity profiles. In this context, the increasing understanding of

the biology of MM has resulted in the identification of mul-

tiple potential targets for antibody-based therapy of MM

(Podar et al, 2009; Richardson et al, 2011; van de Donk et al,

2012).

MDX-1097 is an IgG1/j chimeric antibody specific for

free kappa light chain (jFLC), i.e., light chain not tethered

to Ig heavy chain, and the unique cell surface antigen desig-

nated kappa myeloma antigen (KMA). The variable (V)

region genes of MDX-1097 are derived from the mouse

monoclonal antibody (mAb) mKap (K-121) (Dunn et al,

1996). In previous studies mKap has been shown to be effec-

tive in a xenograft model of MM in severe combined immu-

nodeficient (SCID) mice (Raison et al, 2005) and KMA was

shown to be present on cells derived from B cell lymphopro-

liferative disorders, such as lymphoma (Walker et al, 1985)

and Waldenstr€om macroglobulinaemia (Walker et al, 1985;

Jones et al, 2007).

This study aimed to characterize MDX-1097 with respect

to its cell surface antigen-binding characteristics and effector

functions that could engender anti-MM activity in the clini-

cal setting, in which it might be used alone or in combina-

tion with anti-myeloma agents constituting myeloma

standard therapy. Immunomodulatory drugs (IMiDs),

including lenalidomide, are one of the principle classes of

therapeutic agents currently used to treat MM (Morgan,

2010; Chanan-Khan et al, 2013). Investigations into the

mode of action of IMiDs have shown that lenalidomide

inhibits MM cell proliferation and induces MM cell death

both directly and indirectly via a number of mechanisms,

including the augmentation of Natural Killer (NK) cell acti-

vation and cytotoxicity (Quach et al, 2010).

MDX-1097 has been used in single (Spencer et al, 2010)

and multi-dose clinical trials (Dunn et al, 2013; unpublished

observations) in jMM patients with stable measurable dis-

ease and the results presented here provide a compelling

rationale for further clinical development of MDX-1097 in

combination with established therapeutic agents to improve

clinical outcome in jMM patients.

Materials and methods

Cell culture

KMS-11 and KMS-26 cells were a kind gift from Dr. Takemi

Otsuki of the Kawasaki Medical School, Japan. All other

kappa human myeloma cell lines (jHMCLs) were obtained

from either the American Type Culture Collection (ATCC,

Manassas, VA, USA) or Deutsche Sammlung von Mikroor-

ganismen und Zellkulturen (DSMZ, Braunschweig, Germany)

cell banks.

BM mononuclear cell and peripheral blood mononuclearcell isolation

Bone marrow aspirates and peripheral blood samples from le-

nalidomide-treated jMM patients were obtained, following

patient consent with institutional ethics committee approval,

and MNCs were isolated using density gradient centrifugation.

Similarly, buffy coats or whole blood, obtained from the Aus-

tralian Red Cross Blood Service, with institutional ethics com-

mittee approval, were used to isolate normal PBMCs.

MDX-1097 binding to KMA

Kappa human multiple myeloma cell lines were tested for

KMA expression using APC-MDX1097 in both flow cytome-

try and confocal microscopy experiments. BM MNCs were

stained with APC-MDX1097 (or isotype control). MDX-1097

was produced to current good manufacturing practice

(cGMP) standards by Medarex Inc. (Princeton, NJ, USA)

and labelled, along with its matched isotype, by Invitrogen

(Carlsbad, CA, USA). The cells were simultaneously stained

with anti-CD45-fluorescein isothiocyanate (FITC), anti-

CD138-phycoerythrin (PE and anti-CD38-peridinin chloro-

phyll-cyanin5.5 (PerCP-Cy5.5) and analysed by flow cytome-

try. For inhibition experiments, JJN3 cells were incubated

with biotinylated MDX-1097, alone or in the presence of

hIgG/j or jFLC, and analysed for MDX1097 binding.

Measurement of the affinity of MDX-1097 for jFLC andKMA

The affinity of MDX-1097 for jFLC was determined using

kinetic surface plasmon resonance (SPR). MDX-1097 was

captured on a BiaCore 2000 biosensor chip and jFLC bind-

ing curves were overlayed to calculate rate constants. The

affinity of MDX-1097 for KMA, expressed on JJN3 and

KMS-11 cells, was measured using an adaptation of the

method developed by Bator and Reading (1989). Briefly, cells

were incubated with a range of MDX-1097 concentrations

(1–20 nmol/l) and the amount of un-bound antibody was

measured. Un-bound and bound (total minus unbound)

antibody concentration data was fitted with a non-linear

regression curve (GRAPHPAD PRISM 5; GraphPad Software Inc.,

San Diego, CA, USA) and maximum specific binding (Bmax)

and equilibrium binding constant (Kd) values were calcu-

lated.

Immunohistochemical analysis of MDX-1097 binding

As part of the pre-clinical development of MDX-1097 a

Good Laboratory Practice (GLP) human tissue cross-reactivity

P. Asvadi et al

2 ª 2015 John Wiley & Sons Ltd, British Journal of Haematology

study was conducted by Charles River Laboratories Pathology

Associates (Frederick, MD, USA). Briefly, cryosections from

the BM of patients with j or k PC dyscrasias and a panel

of normal human tissue were stained with FITC-labelled

MDX-1097.

Myeloma drug treatment

JJN3 cells were treated with 1 lmol/l lenalidomide and dexa-

methasone alone or in combination for 5 d. PMBCs were

treated with 1 lmol/l lenalidomide for 3 d. PBMCs were

obtained from 3 MM patients (2 jMM and 1 kMM) prior

to and following 1 month (two patients) or 3 months (one

patient) of lenalidomide (initially 10 mg/d, escalating to

15 mg/d after 2 months) maintenance therapy. In addition

to lenalidomide, the patients received prednisone, 50 mg/d.

Antibody-dependent cellular cytotoxicity assay

Antibody-dependent cellular cytotoxicity (ADCC) was mea-

sured using flow cytometry. Briefly, carboxyfluorescein succ-

inimidyl ester (CFSE)-labelled, MDX-1097 (or isotype

control) coated, target cells were incubated with peripheral

blood mononuclear cell (PBMC) effector cells in a range of

effector to target (E:T) ratios. Alternatively, a range of anti-

body concentrations and a fixed E:T ratio was used. 7-amin-

oactinomycin D (7-AAD) was used to measure cell death. To

elucidate the effect of jFLC on MDX-1097-mediated ADCC,

jFLC was added to the assay mixture and target cell toxicity

was measured.

Results

MDX-1097 binds to KMA on jHMCLs and PCs fromMM patient BM aspirates

Specific KMA binding by MDX-1097 was demonstrated using

flow cytometry, confocal microscopy and immunohistocehm-

istry. In flow cytometry experiments, jHMCLs showed differ-

ential surface expression of KMA (Fig 1A). JJN3, KMS-11 and

KMS-26 cell lines expressed high levels of KMA but no KMA

expression was detected on NCI-H929 cells.

Confocal microscopy experiments were utilized to illus-

trate and confirm the cell surface residence of KMA. In these

experiments, co-staining for KMA and CD59 was carried

out. CD59 was selected on the basis of information support-

ing its constitutive residence in membrane micro-domains

known as rafts. Co-detection of KMA and CD59 was consid-

ered a preliminary step in investigating the molecular inter-

actions at the cell surface that result in generation of KMA.

KMA was detected on the JJN3 cell surface, both in domains

that were positive for CD59 and in isolation (Fig 1B).

In the tissue cross reactivity study, MDX-1097 bound

unfixed BM sections from patients with j but not k isotype-

restricted PC dyscrasias (Fig 1C) or normal BM samples. The

tissue cross reactivity study used a panel of 35 human tissues

obtained from three independent normal donors. No unex-

pected or off-target staining of the normal human tissue

panel was observed (Figure S1). MDX-1097 staining of occa-

sional mononuclear cells in the tonsil was interpreted to be

the result of interaction with anatomically located B or PCs

that produce jFLC. Low-level MDX-1097 staining of intra-

vascular, interstitial and/or leaked proteinaceous material was

deemed to be consistent with binding to residual jFLC in

plasma, serum or tissue (Figure S1).

Analysis of jMM patient BM samples was undertaken to

establish the presence of KMA on primary BM PCs and to

investigate the CD38, CD138 and CD45 status of the

KMA-positive population(s). Figure 2 shows representative

flow cytometry profiles from two patients (Patients 18 and

19, Table I). In Fig 2A, the majority of CD38+ cells are

CD138-negative, however, a proportion of cells are CD138+and both these populations are CD45+ as well as KMA+.In addition KMA was detected on CD38+CD138+CD45�cells. In Fig 2B, within the CD38+ population, both

CD138� and CD138++ populations are positive for KMA

(in this BM sample both the CD38+/CD138�/CD45+ and

CD38+/CD138�/CD45� populations were KMA-positive).

The results of the phenotypic analysis of KMA-positive cells

from primary BM samples of 15 patients (Patients 5–19)are summarized in Table I. KMA was detected on samples

from four patients (1–4) for which no CD38/CD138/CD45

information was available. In total, KMA was detected in

17 out of 19 (89%) samples. In one patient sample

(Patient 11), lack of KMA detection was attributed to the

very low proportion of PCs (1%). This data confirmed that

KMA is present on malignant PCs with a variety of pheno-

types.

MDX-1097 preferentially binds KMA over soluble jFLC

Given that MDX-1097 is specific for KMA as well as soluble

jFLC, we evaluated whether the binding of MDX-1097 to

KMA was influenced by the presence of jFLC, either spiked,at specific concentrations, into the assay mixture or as a

component of serum derived from jMM patients. Only

jFLC, and not IgG/j, partially inhibited the binding of

MDX-1097 to JJN3 and KMS-11 cells, reiterating the jFLCspecificity of MDX-1097 (Fig 3A). Significantly higher molar

ratios of jFLC to MDX-1097 (24:1) were needed to promote

this effect and it was demonstrated that the affinity of MDX-

1097 for jFLC was 20 nmol/l, whereas it was 4 nmol/l for

KMA, i.e., five times higher (Fig 3B,C respectively). We

therefore concluded that the relative lack of inhibition of

KMA binding was due to the higher affinity of MDX-1097

for KMA compared to jFLC.The jFLC used to generate this data was purified mono-

clonal Bence Jones protein (BJP). The effect of jFLC on

MDX-1097 binding to KMA utilizing sera from jMM

patients was also investigated. This confirmed the preferential

MDX-1097 Induces Cytotoxicity Against jMM Cells

ª 2015 John Wiley & Sons Ltd, British Journal of Haematology 3

binding of MDX-1097 to KMA in the presence of jFLC.More specifically, the presence of jFLC had a minimal effect

on KMA binding by MDX-1097 with a modest reduction in

the signal derived from MDX-1097-APC binding as the con-

centration of was jFLC increased (Figure S2). Moreover, the

signal registered in the assay was dependent on both the

(B)

II.

III.

IV. KMS-11

NCI-H929

RPMI-8266

K562

JJN3 (A)

KMS-26

I. II. (C)

Log fluorescence

Cou

nt

I.

Fig 1. KMA is expressed on the cell surface of

jhuman myeloma cell lines and on j plasma

cell dyscrasia bone marrow cryosections. (A)

Cells were stained with MDX-1097-APC (open

histogram) or isotype control (solid histogram)

and analysed under the live cell gate. (B) JJN3

cells were co-stained with MDX-1097 and anti-

CD59 antibodies, visualized under a confocal

laser microscope and images acquired. (I)

Selected field of view; merge of green and red

fluorescence [CD59 and j myeloma antigen

(KMA), respectively]. (II) single cell: CD59.

(III) single cell: KMA.(IV) single cell; CD59

and KMA fluorescence merged. (C) MDX-1097

binds (red staining) to j (I) but not to k(II) plasma cell dyscrasia bone marrow

cryosections.

Table I. Phenotype of KMA positive BM PCs.

Patient M protein isotype [M protein] (g/l) [FLC] (mg/l) %PC KMA KMA+ cell phenotype

1 G NA NA NA + NA

2 G 5 16�7 2 + NA

3 G ND 92 6 + NA

4 NA ND 43�1 15* ND ND

5 A ND >4375 30 + CD38++CD138�CD45+

6 A 31 0�18 40 + CD38+CD138+CD45�7 G 16 NA 16 + CD38+CD138+CD45�8 G 58 472 53 + CD38+CD138+CD45�9 M 20 >4375 36 + CD38+CD138+CD45�10 G 4 367�4 27 + CD38++CD138++CD45�11 G 10 65�4 1 ND ND

12 A 7 14�6 32 + CD38+CD138+CD45�13 G 13 6�8 2 + CD38++CD138++CD45+

CD38++CD138++CD45�14 NA NA NA NA + CD38+CD138+CD45+

15 G 5 5�6 3 + CD38+CD138�CD45+

16 G 4 7�8 2 + CD38+CD138�CD45+

17 G 11 19�9 35 + CD38++CD138++CD45+

CD38++CD138++CD45�CD38+CD138�CD45+

18 NA NA NA NA + CD38+CD138+CD45+

CD38+CD138�CD45+

19 G 32 1123�8 45 + CD38+CD138+CD45+

CD38+CD138+CD45�CD38+CD138�CD45+

CD38+CD138�CD45�

KMA, j myeloma antigen; BM, bone marrow; PC, plasma cell; FLC, free light chain; NA, not available; ND, not detected.

M protein isotype and concentration, FLC concentration and proportion of BM plasma cells (%PC) when BM aspirates were obtained are shown.

The fluorescence-activated cell sorting profile of Patients 18 and 19 are shown in Fig 2.

*The phenotype of the PCs in sample 4 was CD38+CD138+CD45�.

P. Asvadi et al


jFLC concentration of the sera and the concentration of

MDX-1097-APC indicating a dynamic interplay between the

concentrations of MDX-1097 and jFLC with respect to

KMA binding.

MDX-1097 mediates ADCC of jHMCL cells

Antibody-dependent cellular cytotoxicity occurs when Fc

receptor (FcR)-bearing immune effector cells and antibody-

coated target cells are bridged, resulting in the release of

cytotoxic enzymes from the effector cells. ADCC assays were

done using both healthy donor PBMCs and NK cells as

effector cells. When PBMCs were used as effector cells,

MDX-1097 treated JJN3 cells were susceptible to PBMC-

affected ADCC (Fig 4). Furthermore, this effect was recapitu-

lated using alternative KMA expressing cells (KMS-26 and

KMS-11) (Figure S3). Similar experimental set ups and a

different target cell lysis measurement (lactate dehydrogenase

release assay) produced comparable results (Figure S4).

When soluble jFLC was included in ADCC assay mixtures, a

modest dose-dependent reduction in target cell toxicity was

observed (Figure S5).

In order to dissect the contribution of different immune

effector cell populations to MDX-1097-mediated ADCC,

(B)(B)

(A)(A)

Fig 2. KMA is present on plasma cells from j-isotype restricted multiple myeloma patients manifesting divergent CD45 and CD138 expression.

Bone marrow mononuclear cells were stained with anti-CD45, anti-CD138, anti-CD38 and MDX-1097 antibodies. (A) The majority of live cells

(panel I: gate shown; FSC: Forward scatter; SSC: side scatter) are CD38+CD138- (panel II: right lower quadrant) and CD45+ (panel IV; right

upper quadrant) while a small number of cells are CD38+CD138+CD45+ (right upper quadrant, panel II and III). Both populations are j mye-

loma antigen (KMA) positive (histogram overlays on the right) as shown by a shift of MDX-1097 fluorescent peak (blue) compared to the isotype

control peak (red). (B) The majority of the live cells (panel I, gate shown) are CD38+CD138� (panel II; lower right quadrant) while a distinct

CD38++CD138++ is also detected (panel II; upper right quadrant). In the CD38+/CD138- population, the majority of cells are CD45+ (panel IV:

upper right quadrant) while in the CD38++CD138++ the majority of cells are CD45� (panel III; lower right quadrant). Both above populations

also contain CD45� and CD45+ cells (panel III: upper right quadrant and panel IV lower right quadrant). All populations detected are KMA-

positive.



purified NK cells or monoctyes were used as effector cells.

This demonstrated that NK cells consistently elicited ADCC

(Figure S6) whereas the ADCC levels elicited by monocytes

were variable and, in some instances, negligible (data not

shown).

Lenalidomide increases KMA expression and both invitro and in vivo lenalidomide-exposed PBMCs elicitenhanced MDX-1097-mediated ADCC

In vitro experiments were conducted to simulate the clinical

usage of lenalidomide, often in combination with dexameth-

asone, and its effects on KMA and other relevant PC mark-

ers. It was shown that lenalidomide exposure resulted in

expression of statistically significant higher levels of KMA.

Dexamethasone treatment decreased KMA expression levels,

however, when compared to vehicle-treated cells, KMA levels

remained higher in the lenalidomide + dexamethasone-trea-

ted cells (Fig 5A).

The effects on CD38 expression mimicked the effects on

KMA expression, although CD38 expression post-lenalido-

mide + dexamethasone treatment was higher than KMA

expression levels. CD138 expression levels were reduced by

lenalidomide, dexamethasone or their combination.

When in vitro lenalidomide-exposed JJN3 cells were used

in ADCC assays, a higher level of MDX-1097-mediated cell

death was observed (Fig 5B). This increase in cytotoxicity

could be attributed to the increase in KMA levels enabling

increased MDX-1097 binding and therefore more potent

immune effector cell engagement. Similarly, in vitro lenalido-

mide treatment of effector PBMCs resulted in higher levels

of MDX-1097-mediated ADCC when compared to that seen

with vehicle treated PBMCs (Fig 5C).

Importantly, given that lenalidomide is currently used for

MM treatment, we investigated whether in vivo lenalido-

mide-exposed effector PBMCs from MM patients could elicit

enhanced MDX-1097-mediated cytotoxicity of JJN3 cells. It

was shown that PBMCs isolated from MM patients prior to

lenalidomide therapy elicited MDX-1097-mediated ADCC of

JJN3 cells at levels comparable to PBMCs obtained from nor-

mal donors, whereas PBMCs obtained from the same

patients subsequent to initiating therapy with lenalidomide

elicited significantly higher levels of MDX-1097-mediated

ADCC (Fig 6). Notably, these patients were receiving, in

addition to lenalidomide, a daily dose of prednisone. There-

fore, it could be concluded that the in vivo presence of pred-

nisone had not altered the immune potentiating effects of

lenalidomide.

Discussion

The goal of antibody therapy for cancer is the specific target-

ing of malignant cells or the cellular processes that contribute

to their survival and/or drug resistance. Here we have

defined the functional characteristics of the chimeric mAb

MDX-1097 and demonstrated the clear therapeutic potential

of the antibody for the treatment of MM. Data is presented

on the specific interaction of MDX-1097 with KMA on BM

Cou

nt

KMS-11 C

ount

JJN3

MDX-1097 affinity for κFLC

KA (1/M) KD (nM) Chi2

Fc3 5·09 x 107 19·6 19·7

Fc4 4·85 x 107 20·6 4·1

Mean 4·97 x 107 20·1

MDX-1097 affinity for KMABmax (nM) KD (nM)

Mean SD Mean SD

JJN3 2·4 1·1 3·8 1·3 KMS-11 2·9 3·9 4·3 3·2

Mean 2·6 4·0

Log fluorescence Log fluorescence

(A)

(B)

(C)

Fig 3. MDX1097 has higher affinity for KMA

than soluble jFLC. (A) Cells were stained with

biotinylated MDX-1097 either alone, or in the

presence of IgG/j or jFLC, and MDX-1097

binding was quantified. Peaks: black: cells only,

grey: detection antibody only, red: MDX-1097,

blue: MDX-1097+IgG/j, green: MDX-

1097+jFLC. (B) The BiaCore Fc (flow channel)

3 and 4 had high and low levels of antibody

immobilized. (C) After incubation with cells

(as shown) the unbound and bound concentra-

tions were used to estimate the Bmax and kd

values. Three independent experiments were

conducted using the 2 cell lines. KMA, j mye-

loma antigen; jFLC, free j light chain; KA,

association constant; KD, affinity constant; chi-

square, ‘goodness of fit’ measure; Bmax, maxi-

mum specific binding.

P. Asvadi et al


PCs from jMM patients and the absence of KMA on normal

human tissue or other immune cells. The tissue cross reactiv-

ity study reported here identified rare tonsillar cells that

express KMA. Similarly, Hutchinson et al (2014) reported

locating KMA-positive cells in human tonsils. Comparison of

the number of KMA-positive cells located in human tonsils

(Figure S1) and the BM (Fig 1C) clearly indicates the rarity

of tonsillar KMA-positive cells and the abundance of BM

KMA-positive PCs. This suggests that in a clinical setting

MDX-1097 would have a high degree of specificity against

BM PCs, thus promoting a favourable efficacy and toxicity

profile. The significance of the CD45 phenotype in MM

stratification, disease stage, response to therapy and progno-

sis has been the subject of a number of studies (Joshua et al,

1996; Medina et al, 2002; Moreau et al, 2004; Kumar et al,

2005; Ishikawa et al, 2006). Furthermore, the CD138-negative

phenotype has been suggested to be a marker of MM pro-

genitor cells and as a factor affecting response to therapy

(Matsui et al, 2008; Kawano et al, 2012). Therefore, the fact

that MDX-1097 clearly binds to MM PCs expressing varia-

tions of both CD45 and CD138 may provide an important

advantage in the clinical utility of the antibody.

Characterization of the MDX-1097 epitope on jFLC has

provided evidence regarding the conformational nature of the

epitope and a structurally based rationale as to why this epi-

tope is not accessible when jLC is coupled to an Ig heavy chain

(Hutchinson et al, 2011). In the confocal microscopy experi-

ments reported here, the detection of KMA in foci positive for

CD59 suggests partial residence of KMA in raft cellular subdo-

mains (Suzuki et al, 2012), which are rich in sphingomyelin

and cholesterol (Brown & London, 2000) and believed to be

involved in the organization of various signal transduction

pathways (Simons & Toomre, 2000; Gao et al, 2011). This par-

tial raft residence hypothesis correlates with the studies con-

ducted by Hutchinson et al (2010) that provide substantial

evidence for lipid (sphingomyelin)-protein (jFLC) interac-

tions. Importantly, in our study, while the serum jFLC con-

centrations of patients studied ranged from 0�18 to >4375 mg/l

(Table I), KMA was detected in all but 2 instances (with serum

jFLC concentrations of 43�1 and 65�4 mg/l). This indicates

that the absolute level of soluble jFLC is not a major determi-

nant of the quantity of cell surface KMA and suggests the

involvement of alternative cellular mechanism(s) in retaining

jFLC in the cellular membrane, thereby creating KMA.

In addition to binding to KMA, MDX-1097 binds soluble

jFLC and an elevated level of serum FLC is a feature of MM.

Importantly, however, our studies indicate that binding of

MDX-1097 to KMA is not abrogated to any significant degree

by soluble jFLC and provide a rationale, i.e., the higher affin-

ity of MDX-1097 for KMA, for this occurrence. Considering

that in the great majority of MM patients the serum jFLC con-

centrations are below 200 mg/l (Mead et al, 2004), it could be

proposed that, in the clinical setting, the presence of soluble

jFLC binding will not diminish the clinical potential of MDX-

1097 when targeting KMA bearing MM cells. This proposal is

also supported by the results from ADCC experiments in

which jFLC were present in the assay mixture.

Binding of MDX-1097 to serum jFLC may have the poten-

tial for immune complex formation in vivo. However, previous

studies with MDX-1097’s parent mAb (K121, mKap) indicate

that, due to the existence of a single epitope on jFLC, onlycomplexes with an overall composition of two antibody mole-

cules bound to two jFLC monomers (180kD) or two jFLCdimers (400 kD) were formed (Raison & Boux, 1985). Typi-

cally, serum jFLC exists as monomers, therefore it is unlikely

that antibody-antigen complexes >400 kD would be formed.

In addition, size exclusion chromatography studies using

MDX-1097 have demonstrated that even in the presence of

(A)

(B)

Fig 4. MDX-1097 mediates antibody-dependent cellular cytotoxicity

of KMA-expressing jHMCL cells. (A) Labelled JJN3 cells were trea-

ted with MDX-1097 (black bars) or isotype control (white bars)

before the addition of peripheral blood mononuclear cells (PBMCs).

Data represents the average of six independent experiments � stan-

dard error of the mean. Statistical significance (P value <0�05) is rep-resented with one asterisk. (B) Labelled JJN3 cells were incubated

with different concentrations of MDX-1097 (black bars) or isotype

control (white bars) before adding PBMCs at a fixed 100:1 Effector:

Target ratio. Statistical significance is represented with two asterisks

(P value <0�001) or one asterisk (P value <0�05). KMA, j myeloma

antigen.



excess jFLC, MDX-1097 forms an antibody-antigen complex

of c. 180–200 kD (data not shown). Finally, data from the

phase I and IIa clinical trials of MDX-1097 (Spencer et al,

2010; Dunn et al, 2013) in jMM patients support these in vitro

observations as there was no evidence of immune complex for-

mation or organ damage.

MDX-1097 was shown to mediate ADCC, which is consid-

ered to be the major mechanism of action elicited by a range

of therapeutic antibodies in various haematological malignan-

cies (Cooley et al, 1999; Hayashi et al, 2003; Golay et al, 2006;

van Meerten et al, 2006; Tai et al, 2008; Taylor et al, 2009).

MDX-1097-mediated ADCC activity against MM cells was

comparable to other anti-MM antibodies undergoing develop-

ment, for example the anti-CD40 (Hayashi et al, 2003) and the

anti-CS1 (elotuzumb) (Tai et al, 2008) mAbs. NK cells, but

not monocytes, were capable of inducing ADCC against cells

which expressed KMA with the difference in the ADCC-induc-

ing capacity of NK cells and monocytes attributed to the diver-

gence of the range of Fc receptors (FcRs) expressed by the two

cell populations (Nimmerjahn & Ravetch, 2008).

(A)

(B)

(C)

Fig 5. In vitro lenalidomide exposure enhances

KMA expression levels and MDX-1097-medi-

ated ADCC. (A) Fold increase in the mean flu-

orescence intensity (MFI) of vehicle (dimethyl

sulfoxide, DMSO), lenalidomide (Len), dexa-

methasone (Dex) or Len+Dex-treated JJN3

cells stained for KMA, CD38 and CD138 is

shown. Data was collated from four indepen-

dent experiments (error bars represent stan-

dard error of the mean [SEM]). Two-way

analysis of variance (ANOVA) and Bonferroni

post tests were used to determine statistical sig-

nificance (t-test with P value <0�05; denotedwith asterisks) in expression level changes.

Reduction in the level of CD138 expression

post-Len and -Dex treatment was also signifi-

cant (not represented on the graph). (B) Len-

(black and white bars) or DMSO- (grey and

hatched bars) treated JJN3 cells were used in

antibody-dependent cellular cytotoxicity

(ADCC) assays. Data represent the average of

three independent experiments � SEM. (C)

Len- (black and white bars) or DMSO- (grey

and hatched bars) treated PBMCs were used in

ADCC assays. Data represents the average of

three independent experiments � SEM. Tx,

treatment.

P. Asvadi et al


Lenalidomide treatment of JJN3 cells increased KMA

expression and this translated into increased MDX-1097-

induced ADCC of the JJN3 cells. Lenalidomide treatment of

JJN3 cells also increased CD38 expression levels in agreement

with data characterizing the function of the anti-CD38 anti-

body daratumumab (Endell et al, 2012). The effect of dexa-

methasone on KMA expression was also examined because the

anti-inflammatory and anti-proliferative properties of corti-

costeroids, such as dexamethasone, have made it part of the

therapeutic regimen for MM. Although dexamethasone

decreased KMA expression levels, it did not abrogate the aug-

menting effects of lenalidomide on KMA expression. There-

fore, it can be anticipated that in a clinical setting when

lenalidomide and corticosteroids are used in combination, the

PC targeting of MDX-1097 is not significantly diminished.

Furthermore, the in vitro or in vivo exposure of PBMCs to

lenalidomide significantly potentiated the MDX-1097-mediated

ADCC of KMA-positive target cells. Importantly, the net

result of these two lenalidomide-mediated effects was an incre-

mental increase in MDX1097-induced ADCC of KMA-positive

MM cells which, similar to the case of other mAbs (van der

Veer et al, 2011), is attributed to the potentiation of NK cell

function by lenalidomide. Importantly, in the case of our in

vivo results, the significant potentiation of NK cell capacity to

elicit MDX-1097-mediated ADCC activity is present even

when patients were receiving corticosteroid therapy. Although

certain immune-potentiating effects of IMiDs on NK and T

cells have been reported to be reduced by dexamethasone

(Gandhi et al, 2010; Hsu et al, 2011), our data indicates that

MDX-1097-mediated ADCC activity remains significantly

augmented after exposure to both IMiDs and corticosteroids.

In summary, MDX-1097 specifically binds KMA on the

surface of jHMCLs and primary jMM cells and induces

ADCC against KMA bearing cells. Moreover, lenalidomide

treatment of both immune effector and target cells, the latter

via up-regulation of KMA expression, promotes the synergis-

tic killing of KMA expressing MM cells. These data provide a

compelling rationale for the implementation of clinical trials

in j-isotype restricted MM, incorporating a combination of

lenalidomide and MDX-1097.

Acknowledgements

The authors would like to thank the medical and nursing

staff of the Alfred Hospital for assistance in obtaining pri-

mary tumour samples.

Authorship and disclosures

PA designed and conducted experiments, analysed data,

reviewed results and wrote the manuscript. AC designed and

conducted experiments, analysed data, reviewed results and

critically evaluated the manuscript. RD designed experiments,

analysed data, reviewed results and critically evaluated this

manuscript. AS designed experiments, reviewed results and

edited the manuscript. VJ, MW and DJ performed experi-

ments and collated data. TK assisted in planning experi-

ments. All authors reviewed the manuscript. PA, RD and VJ

are current, and MW and DJ are former employees of

Immune System Therapeutics (IST) Ltd. RD owns IST

shares. PA, VJ, MW and DJ as well as AC, TK and AS have

no conflict of interest to disclose.

Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Data S1. Materials and methods.

Fig S1. MDX-1097 shows no off-target binding in a

human tissue cross-reactivity study.

Fig 6. PBMCs exposed to lenalidomide in vivo induce a significantly higher level of MDX-1097-mediated antibody-dependent cellular cytotoxicity

than lenalidomide-na€ıve PBMC. PBMCs obtained from patients prior to (black and grey bars) or after lenalidomide treatment (white and hatched

bars) were mixed at different ratios with labelled JJN3 cells coated with 200 lg/ml MDX-1097 (black and white bars) or human IgG isotype (grey

and hatched bars). % JJN3 cell death was determined as described. Data represents the average of three patients � standard error of the mean.

**: P < 0�001, *: P < 0�05 as determined by 2-way analysis of variance. PMBC, peripheral blood mononuclear cells.



Fig S2. MDX-1097 binds JJN3 jHMCL cells in the pres-

ence of soluble jFLC.Fig S3. MDX-1097 mediates toxicity against KMS-26 and

KMS-11 jHMCLs.

Fig S4. MDX-1097 mediates ADCC against JJN3 target as

assessed using the LDH release assay.

Fig S5. Effects of kFLC on MDX-1097 mediated ADCC.

Fig S6. MDX-1097 mediates NK cell toxicity against JJN3

cells.

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