A Phase II Study of Anti-CD3 x Anti-HER2/Neu (Her2Bi) Armed Activated T Cells (ATC) After Neoadjuvant Chemotherapy in Women With HER2/Neu (0-2+), Hormone Receptor (HR) Negative Stage II-III Breast Cancers
Phase: PHASE2
Conditions: Breast Cancer
Sponsor: Barbara Ann Karmanos Cancer Institute

V05/12/15 
1
Version:  05/10/10, 08/19/10, 1/3/11; 5/23/11, 10/11/11, 08/27/12, 09/03/13, 1/21/2014, 05/07/14, 
0909/14, 03/20/2015, 05/12/2015 
 
Breast Cancer Program 
BMT and Immunotherapy Program 
Barbara Ann Karmanos Cancer Institute 
 
2010-056:  A Phase II Study of Anti-CD3 x Anti-HER2/neu (Her2Bi) Armed Activated T 
Cells (ATC) after Neoadjuvant Chemotherapy in Patients with HER2/neu (0-2+)-Negative 
Stage II-III Breast Cancers 
 
 
IND 9985 
 
PRINCIPAL INVESTIGATOR 
Amy Weise, D.O.  
Barbara Ann Karmanos Cancer Institute 
4100 John R. Mailcode: HW04H0 
Detroit, MI 48201 
 
CO-PRINCIPAL INVESTIGATORS 
Lawrence G. Lum, M.D., D.Sc. 
Abhinav Deol, M.D. 
Archana Thakur, Ph.D. 
 
CO-INVESTIGATORS 
Lydia Choi, MD 
Larry Flaherty, M.D. 
Michael Simon, M.D. 
Sayeh Lavasani, M.D. 
Rouba Ali-Fehmi, M.D. 
Neb Duric, Ph.D. 
David Gorski, M.D., Ph.D. 
 
Biostatistics 
Judith Abrams, Ph.D. 
 
CLINICAL COORDINATORS 
 
Kristie Fields, RN, BSN, OCN 
313-576-8384 
Fax 313-576-8424 
 
 
DATA MANAGEMENT 
Danielle Smith  
Phone 313-576--8506 
Fax 313-576-9212 
 
 
 
 
 

V05/12/15 
2
SUMMARY 
Women with breast cancer who achieve a complete pathologic response (pCR) after preoperative 
(neoadjuvant) chemotherapy (chemoT) have a better prognosis than to those with residual disease; pCR 
is a good surrogate of long-term survival and cure from breast cancer. Women with stage II-III operable 
triple negative breast cancer with residual disease at the time of surgery are  the focus population of this 
protocol.  
Many studies have addressed how to improve the efficacy of chemoT and increase the pCR rate by 
including different chemotherapeutic agents, biologic agents, regimens and schedules. Up to now, apart 
from anti-Her2 targeting antibodies which are highly effective for HER2-positive breast cancer, no 
targeted, non-hormonal agent can be considered a standard therapeutic option for primary systemic 
treatment. We are proposing a novel combination of Immunotherapy (IT) in addition to neoadjuvant 
chemoT for women with stage II-III operable triple-negative breast cancer who have residual disease at 
surgery. We will conduct a phase II trial to evaluate whether infusions of anti-CD3 x anti-Her2/neu 
bispecific antibody (HER2Bi)-armed activated T cells (ATC) after neoadjuvant chemoT, surgery, and/or 
radiation will improve time to progression (PFS) beyond previously published clinical trials. 
Low or moderate expression of HER2 may be targeted by receptor-specific therapies such as targeted 
ATC. This, indeed, is the case in our preclinical and phase I studies in which we used an 
immunotherapeutic strategy to target low to high level expression Her2/neu breast cancer. Evidence of 
clinical and immunologic responses in women with HER2 0/1+ status in our phase I trials would 
suggest a therapeutic benefit even in the absence of HER2 over-expression and provides a 
rationale for studying IT with armed ATC in HER2-negative patients. More importantly, IT after 
treatment (neoadjuvant chemotherapy, surgery, and/or radiotherapy) may have several clinical 
advantages including: (1) removal of tumor with only microscopic minimal residual disease remaining prior 
to IT; (2) the reversal of tumor-induced suppression of immune-based regulatory factors which limits the 
effectiveness of vaccine-induced, tumor-specific T cells in the tumor microenvironment. Following 
Leukapheresis the T-cells will - be activated with anti-CD3 and expanded in 100 IU/ml of IL-2 to generate 
ATC. After culture, ATC will be harvested, armed with HER2Bi, washed, and cryopreserved in 8 aliquots 
for two infusions per week for 4 weeks in combination with IL-2 and GM-CSF.  Patients will be allowed to 
have received anthracycline and/or taxane based standard regimens.  Following chemoT, surgery, and/or 
radiation, patients will be evaluated for chemoT, surgical, and radiation-related toxicities, and then given 
~10-15 billion Her2Bi-armed ATC twice per week for 4 weeks beginning no sooner than 3 weeks (± 1 
week) after the breast cancer treatment. In the phase I trial: (1) the maximum tolerated dose (MTD) of 
HER2Bi-armed ATC was not reached with no dose-limiting toxicities using ~20 billion HER2Bi-armed 
ATC/infusion; and (2) there was development of cytotoxic T cell responses to breast cancer cell lines with 
increases in IL-2, IFNγ, TNFα, GM-CSF, and IL-12.  Therefore, we selected a dose of ~10-15 billion 
HER2Bi-armed ATC per infusion. Armed ATC will be thawed and infused twice a week for 4 weeks for a 
total dose of~80-120 billion armed ATC in combination with low dose IL-2 and GM-CSF.   
 
 
 
 
 
 

V05/12/15 
3
SCHEMA 
 

V05/12/15 
4
                                            TABLE OF CONTENTS                                                       PAGE 
 
 
Summary……………………………………………………………………………......        2 
 
1. 
Objectives………………………………………………………………………………  
 5 
 
2. 
Background and Rationale……………………………………………………………. 
 5 
 
3 
Eligibility ……………………………………………………………………………. 
21 
 
4. 
Treatment Plan…………………………………………………………………………. 
24 
 
5. 
Potential Toxicity, Dose Modifications, and Management…………………………. 
26 
 
6. 
Drug Formulation……………………………………………………………………... 
27 
 
7. 
Preparation of Armed Activated T Cells……………………………………………… 
28 
 
8. 
Registration and Required Data……………………………………………………… 
28 
 
9. 
Criteria for Evaluation………………………………………………………………… 
30 
 
10. Criteria for Evaluation of Response and Discontinuation of Therapy……………        30 
 
11. Reporting of Adverse Reactions………………………………………………………. 
31 
 
12. Measurement of Changes in Immune Populations …………………………………… 32 
 
13. Tumor Studies and Phenotypic Evaluations……………………………………….         32 
 
14. Statistical Considerations……………………………………………………………… 
33 
 
15. Clinical Safety Monitoring……………………………………………………………. 
34 
 
       16. 
References……………………………………………………………………………. .     35 
 
 

V05/12/15 
5 
1.0  OBJECTIVES 
 
1.1 To estimate PFS in women with stage II-III triple-negative breast cancer without a complete 
pathologic response (cPR) who receive a regimen of neoadjuvant chemoT, surgery, and/or 
irradiation followed by 8 infusions of ~10-15 x 109 Her2Bi-armed ATC (aATC) given twice per 
week for 4 weeks  in combination with IL-2 (300,000 IU/m2/day) and GM-CSF (250 
µg/m2/twice per week) beginning 3 days before the 1st infusion and ending 1 week after the 
last infusion [defined as immunotherapy]. 
 
1.2 To estimate the change from baseline (pre IT) to post-IT in specific cytotoxicity and IFN-γ 
Elispots of lymphocytes in the blood directed at breast cancer cells.  
   
1.3 To investigate if pathologic response and the changes in numbers and proportion of infiltrating 
cells and cancer stem cells in the tumor at the time of surgery are associated with progressive 
disease. 
 
 
2.0  BACKGROUND AND RATIONALE 
 
2.1 Clinical problem of increased relapse in patients who do not achieve a pCR after 
neoadjuvant chemoT.   
In a study by Guarneri et al. the prognostic value of pCR was confirmed in 1,731 patients with breast 
cancer who received neoadjuvant chemoT1.  pCR was defined as complete disappearance of invasive 
carcinoma in both breast and axillary lymph nodes. Ninety-one percent of patients received 
anthracycline-based chemoT and 66% received an additional taxane.  After adjustment for other 
prognostic factors, patients who achieved a pCR had 0.41 times the risk of relapse and 0.36 times the 
risk of death compared with patients who did not achieve a pCR.  Among patients without hormone 
receptor (HR) expression, pCR was achieved in 24% compared to 8% in HR positive patients.   
 
A study by Carey et al. evaluated responses to neoadjuvant chemoT in 107 patients with stage II and 
III breast cancer2.  Patients received neoadjuvant doxorubicin (60 mg/m2) plus cyclophosphamide (600 
mg/m2) chemoT (AC) given i.v. for 4 cycles, either alone or as the first component of a sequential AC-
taxane neoadjuvant regimen. Twenty-eight patients (26%) received AC on a dose-dense schedule 
(every 2 weeks), whereas the rest of the patients received AC on an every 3 weeks schedule.  Most 
patients (80 of 107, 75%) received additional neoadjuvant chemoT following AC, which primarily 
involved either paclitaxel or docetaxel.  pCR to chemoT (defined as post operatively stage 0, no 
invasive cancer) was significantly better among the basal-like subtype (27%), defined as the 
immunohistochemical surrogate ER-negative, PR-negative, and HER2-negative (triple-negative), and 
the HER2-positive/ER-negative subtype (36%) versus the combined luminal subtypes (7%, P = 0.01). 
However, despite initial chemosensitivity, patients with the basal-like and HER2-positive/ER-negative 
subtypes had worse DFS (P = .04) and OS (P = .02) than those with luminal subtypes.   
 
Liedtke et al published an analysis of a prospectively collected clinical database including  1,118 
patients who received neoadjuvant chemoT at M.D. Anderson Cancer Center for stage I-III breast 
cancer from 1985 to 2004 and for whom complete receptor information was available3. The primary 
goal was to describe the relation between triple-negative breast cancer (TNBC) and major determinants 
of clinical outcome, such as response to neoadjuvant chemoT (rate of pCR in breast and lymph nodes 
[pCR]), progression-free survival (PFS), site-specific distribution of recurrence, post recurrence survival 
(PRS), and overall survival (OS). Two hundred fifty-five patients (23%) were designated as having 
TNBC and 863 patients (77%) were designated as non-TNBC. Increased pCR rates were observed for 
patients with TNBC compared with non-TNBC (22% v 11%; odds ratio [OR] = 1.53; 95% CI, 1.03 to 
2.26; P = .034). The highest pCR rates (28%) in association with TNBC were observed for patients 
treated with chemoT regimens containing a taxane (weekly/once every 3 weeks paclitaxel/docetaxel) 
followed by anthracycline-based regimens (fluorouracil, doxorubicin, and cyclophosphamide or 
fluorouracil, epirubicin, and cyclophosphamide). TNBC patients with residual disease after 

V05/12/15 
6 
neoadjuvant chemoT have significantly worse survival, particularly in the first 3 years  
compared with patients with non-TNBC and residual disease  (HR = 1.5; 95% CI, 1.3 to 1.8; P < 
.0001)3. 
 
2.2 Underserved population of women with TNBC.    
The studies cited in the previous section demonstrate the worse prognosis of patients with TNBC, 
especially when they do not achieve pCR.  Since they are not eligible for anti-estrogen (hormonal) 
therapy, there are no other standard treatments beyond the neoadjuvant chemoT and local treatment 
(surgery +/- radiation). Treatments targeting this poor risk group of patients is especially important. Of 
specific relevance to this purpose is that African American women tend to have more TNBC4-6, and at 
our center, 44.32% of the neoadjuvant patients from 1997-2008 (KCI database) were  African American 
women with newly diagnosed localized breast cancer.  
 
2.3 Criteria for pCR.   
Patients with breast cancer who achieve a pCR after neoadjuvant chemoT have an improved prognosis 
compared to those with residual disease; pCR is a reliable surrogate marker for long-term survival and 
cure from breast cancer1;7.  In fact, the achievement of a pCR is one of the most reliable surrogate 
markers predicting long-term outcome and the pathologic staging at surgery following preoperative 
treatment maintains its prognostic significance7-10.  The definition and methods of assessment of pCR 
vary across studies. A more stringent definition includes absence of all invasive and non-invasive tumor 
cells in both breast and axilla,1 while others define pCR as absence of invasive tumor in the breast 
irrespective of the nodal status11;12.  Therefore, the definition and assessment of pCR can influence the 
reported rates independently from the activity of different regimens.  However, regardless of the applied 
criteria, patients who achieve a pCR have better outcomes9;13. 
 
2.4 Chemo-resistant and persistent tumorogenic breast cancer CD44hi/CD24lo, aldehyde 
dehydrogenase activity (ALDH1), and CD133 “cancer stem-like cells” (CSC) after ChemoT.  
Persistence of chemoT resistant breast CSC is the most likely the cause of disease recurrence, 
especially if minimal residual disease is attained with chemoT. Breast CSCs are believed to be 
CD44hi/CD24lo tumor cells resistant to and persisting after chemoT. The results from Li showed that 
treatment increased the percentage of CD44hi/CD24lo cells (mean at baseline vs. 12 weeks after 
neoadjuvant chemoT, 4.7% to 13.6%, P < .001)14. The ALDH1 population, as measured by the 
ALDEFLUOR assay, has been used to identify, characterize, and isolate populations from normal and 
malignant mammary tissue displaying stem cell properties in vitro  and in NOD/SCID xenografts15.  
Subsequently, Ginestier et al showed in a series of 577 breast carcinomas that expression of ALDH1 
correlated with poor prognosis16.   CD133 positivity has also been implicated as a co-marker for CSC in 
inflammatory breast cancers that express a stem cell-like phenotype (CD44hi/CD24lo, ALDH1, and 
CD133)17-19.. 
 
2.5 HER2 as a target.  
HER2 belongs to the epidermal growth factor receptor family of tyrosine kinases.  The HER2 oncogene 
encodes a 185-kDa transmembrane receptor with significant sequence homology to the class I 
receptor of the tyrosine kinase family20.  HER2 is over-expressed in breast, ovarian, lung, gastric, 
oral20, and prostate cancers21-23. The expression of HER2 makes it an ideal target for biological-based 
agents such as Herceptin® (trastuzumab), a humanized mAb that targets HER2, which was approved 
for the treatment of women with HER2-positive breast cancer.  Herceptin® is believed to exert its anti-
tumor effects via interference with receptor-mediated signaling pathways,24 inhibiting cell cycle 
progression by inhibiting the mitogen-activated protein kinase pathway25;26, suppressing the anti-
apoptotic phosphatidylinositol 3-kinase and Akt pathways27;28, and antibody-dependent cellular 
cytotoxicity (ADCC)29-33.  Although Herceptin® is effective as a single agent34;35 or in combination with 
cytotoxic chemoT36;37, data from efficacy trials suggest that the beneficial treatment effects are limited 
to patients with the highest levels of HER2 expression.  This limits the application of Herceptin® to 20-
25% of patients with Her2/neu 3+ or fluorescence in situ (FISH) amplified disease, leaving 75-80% of 
the Her2/neu 0-2+, FISH-negative patients without antibody-targeted treatment options. 

V05/12/15 
7 
 
2.6 Targeting low level Her2/neu receptor expression.  
In view of the inherent low level expression of Her2/neu receptors on most breast cancers and the 
potential for the currently available HER2-targeted therapies to cause increased dimerization and 
downregulation of HER2 receptors38, leaving fewer receptor targets on the cell surface for drugs 
designed to target these tumors, a different strategy is required for HER2 non-over-expressing tumors. 
In such instances, very low or nearly absent expression of HER2 may still be targeted by receptor-
specific therapies such as targeted ATC.  This is the case with our preclinical and phase I studies in 
which we used Her2Bi armed T cells to target very low to high level expression Her2/neu breast 
cancer.  Evidence of clinical and immunologic responses in women with HER2 0/1+ status in our 
phase I trials would suggest a therapeutic benefit even in the absence of HER2 over-expression 
and provides encouraging results in the HER2-negative  patients with armed ATC39;40. 
 
2.7 Combination neoadjuvant chemoT and immunotherapy (IT) in TNBC patients who fail to 
achieve pCR.   Many studies have addressed how to improve the efficacy of chemoT and increase the 
pCR rate by evaluating different anthracycline-based regimens, anthracycline–taxane combinations, 
sequential regimens, and dose-dense schedules41-75. Taxane (paclitaxel or docetaxel) and 
anthracycline–based regimens appear to provide higher pCR rates, but the pCR rate is still lower than 
is optimal.  Up to now, apart from trastuzumab for HER2-positive breast cancer, none of the targeted 
agents can be considered a standard therapy for curable disease. The combination of chemoT followed 
by IT may have several advantages including: (1) tumor debulking; and (2) the reversal of tumor-
induced suppression of immune-based regulatory factors which limits the effectiveness of vaccine-
induced, tumor-specific T cells in the tumor microenviroment76. Induction of lymphopenia leads to 
increased availability of IL-7 and IL-15, which enhance CD8+ T cell activity77;78, as well as depletion of 
regulatory T cells from the tumor microenvironment. Other studies have shown enhanced T cell 
trafficking into tumors and intratumoral proliferation of effector cells after induction of lymphopenia.77-79 
Vaccination during homeostasis-driven proliferation may educate the developing T cell repertoire and 
lead to enhanced T cell memory against tumor antigens80;81. Adoptive transfer of naïve or activated 
antigen-specific T cells after induction of lymphopenia has been reported to induce tumor 
regression82;83;84. The most effective anti-tumor immunity is induced when vaccination and 
reconstitution are performed concomitantly82. 
  
In cancer patients, Dudley et al85 demonstrated that lymphodepletion with the combination of 
cyclophosphamide and fludarabine, followed by infusion of ex vivo expanded tumor-specific T cells,  
mediated a significant tumor regression in heavily pretreated patients with IL-2 refractory metastatic 
melanoma.  Recently, studies showed that paclitaxel enhances IL-12 production in tumor-bearing 
hosts86, enhances cytotoxicity of natural killers cells against breast cancer by increasing perforin 
production87, and preferentially impairs regulatory T cells rather than effector T cells88.  Machiels et al 89 
demonstrated in an animal model that paclitaxel, when given one day before vaccine, depleted 
regulatory T cells from the tumor microenvironment and enhanced the vaccine efficacy in Her2/neu 
tolerized mice. In this protocol, we will target patients with operable, triple-negative breast 
cancer and we propose a strategy that will combine the debulking and immune modulating 
effects of neoadjuvant chemoT, surgery, and/or radiation with adoptively transferred armed 
HER2Bi ATC after treatment.  “Third generation” regimens, which are expected to have similar pCR 
rates and are the most aggressive neoadjuvant standard chemoT regimens are recommended, 
allowing patients to have the best available standard treatment in addition to the IT.  These regimens 
all consist of doxorubicin (A), cyclophosphamide (C), and a taxane (T).  The specific regimens will be: 
(1) dose dense ACT [A 60mg/m2 with C 600 mg/m2 every 2 weeks for 4 doses (required with G-CSF 
support), followed by either paclitaxel 175 mg/ m2 every 2 weeks for 4 doses (required with G-CSF 
support) or paclitaxel 80 mg/m2 weekly for 12 doses] or (2) TAC [docetaxel 75 mg/m2, A 50 mg/m2, and 
C 500 mg/m2 in combination every 3 weeks for a total of 6 doses (required with G-CSF support)]. 
 
 2.8 Infusions of BiAb alone in other clinical trials for solid tumors.   

V05/12/15 
8 
In a clinical trial involving 27 patients with breast cancer, infusions of 2B1 BiAb (targeting c-erbB-2 and 
FcγRIII to arm Fc-receptor bearing cells in vivo) alone led to 2 partial responses (PRs) and 3 minor 
responses90. The BiAb MDX-H210 (anti-CD64 x HER-2), was used to treat women with breast 
cancer91.  There was 1 PR and one mixed response in 10 evaluable breast cancer patients.  Using a 
similar approach, several groups have developed second and third generation humanized BiAbs 
reactive to T cells and HER-2 positive tumors92-96.  In a recent phase II advanced prostate and renal 
cancer trial using MDX-H210 in combination with GM-CSF, 5 objective responses (>50% decline in 
PSA) were documented97.  It is likely that armed ATC may provide a similar or better clinical result.  
 
2.9 ATC as Cytotoxic Vehicles and Cytokine/Chemokine Factories.  
Cross-linking the T cell receptor (TCR) of T cells with anti-CD3 leads to activation, proliferation, 
cytokine synthesis [interferon-γ(IFNγ), tumor necrosis factor-α(TNFα),and GM-CSF]98-108, and non-MHC 
restricted cytotoxicity directed at tumor targets109-112. Many chemotherapies kill tumor cells via 
apoptosis and apoptotic cells are a good source of antigens113.   While tumor cell apoptosis induced by 
chemoT leads to increased cross-presentation of tumor antigens114;115, there is a lack of priming 
effective anti-tumor immune responses, suggesting that the transition from cross-presentation to cross-
priming needs help115. If ATC can mediate enhanced cytotoxicity directed at cancers after chemoT, 
armed ATC may have greater anti-tumor activity after chemoT.  
 
2.10 ATC armed with HER2Bi for Metastatic Breast Cancer (MBC). In our phase I clinical trial, we 
have administered ATC armed with HER2Bi to women with both HER2-positive and negative MBC to 
determine their safety and the maximum tolerated dose.  Arming ATC with HER2Bi makes every T cell 
into a HER2-specific cytotoxic T lymphocyte (CTL) with the potential to induce high levels of specific 
cytotoxicity that is independent of HER2 receptor-mediated mechanisms and, thus, HER2 
expression levels116.  Evaluation of immune responses in our phase I clinical trial patients suggests 
that infusions of Her2Bi-armed ATC induce robust immune responses regardless of the patient’s HER2 
status.  Median OS is 56 months for the Her2 3+ group, 20 months for the Her2 0-2+ group, and 26 
months for the entire MBC group with median follow-ups of 28, 17, and 21 months, respectively (Fig-
6). There was no statistical difference between them (p = 0.51), suggesting that armed ATC may 
prolong survival independent of HER2/neu status.  Unlike therapy with Herceptin®, which induces 
cellular death through interference with receptor-mediated signaling pathways, low levels of HER2 
receptors can induce immune responses, even in TNBC patients.  Our clinical data shows that immune 
responses were obtained in women with MBC previously treated with taxanes.  Therefore, IT in 
addition to a standard neoadjuvant taxane containing chemoT regimen  may be ideal to provide anti-
tumor activity and impair T regulatory cell activity while improving NK cell activity.  We also expect it 
would provide a measure of lymphodepletion for the expansion of vaccine responsive immune 
elements.  
2.11 Pre-Clinical Studies: Targeting Cancer Cells with Her2Bi-armed ATC. In preclinical studies, 
we showed that Her2Bi, a bispecific antibody created by chemical heteroconjugation of anti-CD3 
(OKT3; Centocor Ortho Biotech) with anti-HER2 (Herceptin®; Genentech) can be used to arm ATC 
which were produced by  activating PBMC with 20 ng/ml of anti-CD3 and expanded in 100-500 IU/ml of 
IL-2 up to 14 days116.  ATC armed with an optimal dose 50 ng of Her2Bi/106 ATC exhibits high levels of 
specific cytotoxicity against HER2-expressing cell lines derived from breast cancer (SK-BR-3, MCF-
7)116, pancreatic cancer (MIA PaCa-2, COLO 356/FG)117, and prostate cancer (LNCaP, DU 145, PC-3) 
lines118.  At effector/target ratios (E/T) from 3.125:1 to 25:1, Her2Bi-armed normal and armed patient 
ATC are significantly more cytotoxic against low or high HER2-expressing cells than unarmed ATC, or 
ATC armed with an irrelevant BiAb such as anti-CD3 x anti-CD20 BiAb.  

V05/12/15 
9 
 
Pertinent to this proposal, Fig-1 shows high levels 
cytotoxicity directed at extremely low-expressing Her2/neu cell 
line MCF-7 which  expresses <1.0 x 104 Her2 receptors /cell. 
This number of receptors on the tumor cell surface is not 
detectable by IHC. Her2Bi-armed ATC secrete high levels of 
Th1 cytokines when they bind to tumor. Redirected non-MHC 
restricted T cell killing via the perforin/granzyme system is the 
mechanism responsible for enhanced killing. In mice, co-
injections of Her2Bi-armed ATC with PC-3 prostate tumor cells 
in a Winn assay completely prevented development of tumors 
and intravenous infusions of Her2Bi-armed ATC significantly 
delayed growth of established PC-3 tumors compared to mice 
that received ATC or vehicle alone (p< 0.001) without any side 
effects118.  
  
2.12 Her2Bi-Armed ATC Target and Kill SUM 1315 cells 
(Her2- negative cell line).  In order to determine whether 
armed ATC could target and lyse a Her2-negative cell line, 
Sum 1315 (a gift from Stephen Ethier) for cytotoxicity was 
evaluated. Sum 1315 is a breast cancer cell line known to be 
negative for Her2/neu by Western blotting (Fig-2a) and flow 
cytometry(Fig-2b). Fig-2c shows specific cytotoxicity of Her2Bi 
armed ATC directed at Sum 1315 and SK-BR-3. Flow 
cytometry showed that Sum 1315 cells 
had a very low mean fluorescent intensity 
(MFI) and only 6.7% were positive. These 
results are consistent with the Western 
blot (Fig-2a).  Goat anti-Her2/neu was 
used to detect Her2/neu receptors on Sum 
1315 and SK-BR-3 cells. Fig-3c shows 
that armed ATC exhibit high levels of 
cytotoxicity directed at Sum 1315 cells. It 
is clear that only a few molecules of 
HER2/neu on their surface are sufficient to 
allow binding and triggering of specific 
cytotoxicity.  This is consistent with the 
observation that as few as 10-30 TCR-
ligand interactions are sufficient to mediate 
T cell killing119. These data and our clinical 
and immunologic data on patients treated 
with Her2Bi armed ATC who were Her2-
negative in our phase I clinical trial provide 
the rationale to target patients with HER2 
(0-2+) expression.  This approach would 
potentially benefit up to 80% of patients 
with BrCA including those with triple 
negative BrCa.  
 
Fig 1:  % Cytotoxicity (51Cr release) of MCF-7 
Mediated by ATC Armed Normal ATC with 
Increasing Arming Doses of Her2Bi.  ATC were 
armed with 0.5, 5.0, and 50 ng/106 ATC and 
plated at E/T of 25:1, 12.5:1, 6.25:1 and 
3.125:1.  Adherent MCF-7 were seeded (40,000 
cell/well in flat-bottomed plates, labelled with 
51Cr, and effectors were added at the indicated 
E/T and 51Cr release measured after 18 hrs. 

V05/12/15 
10 
2.13 Phase I Clinical Trial Patients and Treatment Schema.  We 
performed a phase I dose escalation clinical trial in women with 
stable to slowly progressive HER2 0-3+ MBC to determine the 
maximum tolerated dose (MTD) for Her2Bi-armed ATC. The 
protocol involved 2 infusions per week for 4 weeks with daily IL-2 
and twice weekly GM-CSF beginning 3 days before the first 
infusion and ending 7 days after the last infusions of armed ATC. 
Table 1 shows the characteristics of 19 women enrolled who had a 
median age of 48 years (range: 31-68 years).  All patients were 
enrolled between September 2002 to January 2008. One patient 
was treated twice (twice at dose 
level 2).  Patients were leukapheresed for T cells that were expanded with anti-CD3 and IL-2 for 14 
days. ATC were harvested, armed with Her2Bi, cryopreserved, thawed, and infused at the bedside.  
Patients were accrued to each dose level (Table 2) with dose escalation based on the criteria that 
persistent grade 3 non-hematological toxicity did not occur in 3 of 3 patients.  If 1 of 3 patients at the 
dose level developed persistent grade 3 toxicity, then an additional 3 patients would be added to the 
dose level.  The dose would be escalated if 5 of 6 patients at that dose level did not have a dose 
limiting toxicity (DLT).  If 2 of 6 at the dose level had a DLT, then the maximum tolerated dose (MTD) 
will be the dose below the dose.  
 
2.14 Phenotype and Viability of the Cryopreserved 
Cell Products. Phenotyping and viability was performed 
on cryopreserved products. Table 3 summarizes the 
%CD3, CD4, CD8 and viability of the  
products that were infused into the patients. The mean % 
CD3, CD4, and CD8 were 86.7%, 52.4%, and 34.6%, 
respectively.  The mean viability was 92.2%. 
 
2.15 Cytotoxicity Mediated by Cryopreserved Armed 
and Unarmed ATC from Patients. Specific cytotoxicity 
directed at SKBR-3 was quantitated on cryopreserved 
armed ATC and unarmed ATC products using the method 
previously described116. Fig-3 summarizes the cytotoxicity 
mediated by cryopreserved armed ATC (P aATC) and 
unarmed ATC (P ATC) from the patients and armed ATC 
(P ATC) and unarmed (N ATC) from normal controls.  
Cytotoxicity was directed at SK-BR-3 targets at an E/T of 
12.5:1. Cytotoxicity mediated by P ATC armed with 
irrelevant anti-CD3 x anti-CD20 BiAb at 12.5:1 was 
comparable to unarmed ATC (data not shown). These are 
the product release data for the cell products used for the 
clinical infusions. Mean cytotoxicity (±SD) mediated by 
cryopreserved patients’ Her2Bi-armed ATC (49.32% ± 

V05/12/15 
11 
21.54) was significantly greater (p < 0.0001) than that mediated by cryopreserved patients’ unarmed 
ATC (3.21 ± 3.20) (Fig 3). The Spearman correlation coefficient comparing % cell type to specific 
cytotoxicity was r = -0.55 with a p = 0.03 for CD4.  There was an inverse correlation between in vitro 
cytotoxicity and the proportion of CD4 cells in the expanded ATC product (Spearman correlation 
coefficient, comparing % cell type to specific cytotoxicity) with an r = -0.55 with a p = 0.03 for CD4. 
 
2.16 Cytotoxicity Mediated by Her2Bi-Armed ATC 
Unchanged from Fresh Armed ATC.  To determine 
whether cytotoxicity mediated by armed or unarmed ATC 
was altered by cryopreservation, cytotoxicity of fresh 
aATC, cryopreserved aATC, and ATC that were armed 
after thawed were compared for cytotoxicity (Fig-4). 
These data show that that cryopreservation did not 
alter the ability of armed ATC to mediated specific 
cytotoxicity. There were no statistical differences in 
selected samples that were tested fresh versus frozen.   
 
2.17 
Methodology 
to 
Detect 
Armed 
ATC 
and 
Correlations with Specific Cytotoxicity Mediated by 
Known (Spike) Proportions of Armed ATC in the 
Peripheral Blood Samples. To determine the lower limit 
of sensitivity for detection of armed ATC-mediated 
cytotoxicity, we performed a Her2Bi-armed ATC spiking 
experiment. PBMC were spiked with 5% Her2Bi-armed 
ATC and Miltenyl selection was performed to separate the 
IgG2a+ 
(OKT3-moiety 
of 
the 
BiAB) 
and 
IgG2a- 
populations.  Each population was analyzed by flow 
cytometry to detect cell bearing IgG2a mouse IgG (Fig-
5A). Staining showed 0.87% IgG2a+ cells in the IgG2a-depleted population whereas the positively 
selected population contained 82.53 IgG2a+ cells. In a parallel study, known proportions of Her2Bi-
armed ATC (0-100%) were mixed with PBMC to create a standard curve for detecting IgG2a+ cells 
(Fig-5B). This standard curve could detect as 
little as 0.87% IgG2a+ cells (Fig-5A) in samples 
containing known proportions of Her2Bi-armed 
ATC. These studies established the lower 
detection limit for flow cytometry for IgG2a+ cells.  
 
2.18  Correlations Between Proportions of 
Spiked Armed ATC and Specific Cytotoxicity 
in the Spike Blood Samples.  Cytotoxicity 
assay at an E/T of 25:1 was performed using a 
range of proportions for armed ATC to PBMC 
(Fig-5C). There was no detectable increase in 
cytotoxicity against SK-BR-3 cells over PBMC 
alone in PBMC populations spiked with 0.1% or 
1% Her2Bi armed ATC. These spiking levels are 
above 
the 
lower 
detection 
limit for flow 
cytometry. These are levels comparable to 
circulating armed ATC levels.  Flow cytometry is 
more sensitive than cytotoxicity assay for 
identification 
of 
armed 
ATC 
populations. 
Together, these data show that the cytotoxicity observed in the blood and in the IgG2a- fractions of 
patient PBMC (see Fig-17 in section 3.21) cannot simply be attributed to the few percent of armed 
ATC detected in the blood. These data show that the enhanced specific cytotoxicity in patients’ PBMC 
Fig-4: Shows comparable cytotoxicity directed at SK-BR-3 by  
patient’s fresh aATC (FaATC), cryopreserved aATC (CaATc), and 
cryopreserved ATC that were armed post-thaw (CATaP) The also 
shows cryopreserved ATC (CATC), normal cryopreserved ATC 
(NCaATC), and cyropreserved ATC that were not armed with Her2Bi. 
Fig-3: 
Summarizes 
cytotoxicity 
mediated 
by 
cryopreserved armed patient ATC (PaATc), normal 
armed ATC (N aATC), patient ATC (PATC) and 
normal ATC (NATC) The means ± 1 S.D. are 
indicated by the error bars. All samples were frozen, 
thawed and then tested as part of quality control 
product release. 

V05/12/15 
12 
is due to endogenous cytotoxicity. 
 
2.19 The Kinetics and Survival of Armed ATC in a Patient.  PBMC were obtained pre and post after 
each infusion to determine the proportion of IgG2a+ cells within the 
CD3+ population (Fig-6). The post infusion values show transiently 
elevated numbers of armed ATC (diamonds) in the circulating that 
return to the CD3+ pre baseline (circles) that range from ~1 to 8%.  Up 
to 50% of the circulating T cells were positive for IgG2a after the 
second infusion.      
 
 
2.20 
Detection 
of 
Circulating murine IgG2a+ 
(OKT3+) Cells in 8 Patients 
during Immunotherapy. To 
confirm the survival of IgG2a+ 
cells in additional patients, we 
tested 
an 
additional 
four 
patients (FG60112, FG60121, 
FG60056, and FG60191) for 
circulating 
armed 
ATC 
by 
phenotyping 
the 
peripheral 
blood for mouse IgG2a+ cells 
(Fig-7). 
 
Phenotyping 
the 
peripheral blood obtained prior 
to 
immunotherapy, 
before 
infusions #5 and #8, and 1 wk 
after the last infusion showed 
evidence for accumulation and 
persistence in some patients of  
armed ATC that last up to 1 
weeks after  
the last infusion. The left panel of Fig-7 shows Dose Level 1 and the right panel shows Dose Levels 2 
and 3.  
 
2.21 In Vivo Trafficking of Her2Bi-armed ATC.  Infusions of 111In labelled Her2Bi-armed ATC 
localized to the bone marrow, lung, liver, and spleen within 4 hours of injection (Fig-8 left panel).  By 
24 hours, Her2Bi-armed ATC had cleared from the lungs but persisted in the bone marrow, liver and 
spleen for up to 4 days post-infusion (Fig-8, right panel). Serial measurements of 111Indium labeled 
armed ATC in whole blood (Fig-9) showed that about 50% Her2Bi-armed ATC are cleared from the 
blood within ~30 min. The patient’s blood samples continue to be positive for radioactivity (1.1% of the 
Fig-5A 
Fig-5B 
Fig-5C 
Fig-5: Shows Phenotyping of Miltenyl purified IgG2a+ and IgG2a- populations (Panel A), 
phenotyping for IgG2a+ cells in spiking experiment wherein various proportion of Her2Bi armed 
ATC were added to PBMC to determine the lower limit of sensitivity of detection for IgG2a+ cells 
(Panel B), and cytotoxicity of PBMC spiked with various proportions of Her2Bi-armed ATC.  
IgG2a+ cells were detected by flow cytometry using goat anti-mouse IgG2a-PE to detect the 
OKT3 moiety of OKT3 x Herceptin. 
Fig-6: Shows the Proportion of 
IgG2a+ and IgG2a- CD3+ Cells as a 
Function of Days after the Start of 
Immunotherapy. PBMC were double 
stained with goat PE conjugated anti-
IgG2a mouse and anti-CD3 to detect 
the proportion of CD3+ that were 
IgG2a+ and IgG2a-.  Pre and 2 hour 
post infusion various were obtained.    
Fig-
7 
Fig-7: Armed ATC (IgG2a+ cells) Detected Up to 1 Week Post Infusion.  Four patients in 
Dose Level 1 (left panel) and 4 patients in Dose Levels 2 and 3 (right panel).  Cells were 
detected in whole blood using goat anti-mouse IgG2a+ antibodies to phenotyping the 
circulating cells prior to IT, before infusions 5 and 8 (inf 5 and inf 8) and 1 week after the 
last infusion (1 week post)

V05/12/15 
13 
initial concentration) up to 4 days 
post infusion. It is clear that armed 
ATC persist up to 1 week or more 
post infusion. 
 
2.22 Localization of Her2Bi-armed 
ATC to Breast Cancer Tumors.  
Formalin-fixed, 
paraffin-embedded 
samples were prepared from a 
chest wall nodule excision and a 
sternal tumor biopsy at 1 month and 
1 week post treatment, respectively. 
Anti-CD3 was used to detect human 
T cells and goat anti-mouse Ig was 
used to detect mouse IgG2a using 
an 
overlay 
strategy. 
 
Tissues 
samples 
were 
sectioned, 
deparaffinized, stained with H&E 
and characterized for tumor content by a pathologist. Adjacent 
sections were stained with anti-CD3 to detect T cells using the 
CSA Peroxidase System (DAKO, Carpinteria, CA) after target 
retrieval and endogenous biotin/avidin and peroxidase 
quenching with the CSA Ancillary System (DAKO). Anti-CD3 
antibody (1 g/ml) was diluted in Background Reducing 
Components (CSA Ancillary System) and incubated with 
tissue samples for 30 min at room temperature.  Primary 
antibody was detected by incubating for 15 min with 
biotinylated goat anti-mouse immunoglobulins, and the signal 
was 
amplified 
and 
visualized 
by 
diaminobenzidine 
precipitation at the antigen site. In parallel, adjacent sections 
were stained with biotinylated goat anti-MIg (directed at the 
mouse IgG2a) followed by streptavidin-FITC to detect the 
goat anti-MIg.  Images acquired using fluorescent filters were 
overlayed upon images acquired by light microscopy creating composite images (Fig-10) to evaluate 
co-localization of staining.  
 
2.23 Evaluation of MTD and Toxicities.  
The purpose of this study was to 
determine the MTD of Her2Bi-armed 
ATC and to define the toxicity profile. 
The MTD is defined as the dose below 
the dose at which dose limiting toxicity is 
encountered 
in 
2 
of 
6 
patients.  
Currently, 
the 
highest 
dose 
level 
completed is the 40 x 109 Her2Bi-armed 
ATC per infusion (320 x 109 total dose of 
armed ATC); the MTD has not yet been 
reached.  We have accrued one patient 
at the dose level of 40 x 109 Her2Bi-
armed ATC per infusion (320 x 109 total 
dose).  The most frequent side effect 
was Grade 3 chills.  Grade 3 headaches 
emerged as the second most common 
side effect.  Fewer than 50% of the total 
Fig-9 
Fig-9: Serial Measurements of 111In Labeled 
aATC in the Blood after Injection of 200 x 106  
Decayed.
Fig-10 
Fig-8 
Fig-8:  111Indium Whole Body Scans Show Trafficking of Armed ATC as a Function 
of Time.  Armed ATC (200 x 106) were labeled with Indium and infused IV. Serial 
scans were obtained at 4 hr (left panel), 24 hr (2nd leftmost panel), 48 hr (2nd 
rightmost panel), and 4 days (rightmost panel). 

V05/12/15 
14 
patients enrolled experienced each of the 
remaining symptoms.  Table 4 shows the 
frequency of side effects in the study as a 
function of dose level based upon the NCI 
Immunotherapy Protocol Toxicity Table.  By 
episode per infusion, incidence of chills and 
headache at dose level 1 (8.6% and 3.1%, 
respectively) increased for dose level 2 
(20.8% and 8.3%, respectively) and then 
again at dose level 3 (43.1% and 19.6%, 
respectively). All patients with grade 3 chills 
responded to Demerol.  One patient at dose 
level 
3 
experienced 
headache 
and 
hypertension that failed to respond to 
interventional therapy (grade 4); the patient 
was removed from the study after receiving 3 
infusions for a total of 65.7 x 109 armed ATC. 
The 
patient 
was 
suspected 
of 
brain 
metastasis, but surgical follow-up revealed a 
right frontal subdural hematoma that was 
evacuated without complications. An adverse 
event was reported and the patient was 
removed from the protocol.  Accordingly, 3 
additional patients were added to dose level 
3. None experienced persistent grade 3 
toxicities and no fatalities occurred as a result 
of armed ATC infusions. One patient has 
received dose level 4 (40 billion/dose) 
successfully.   
2.24 Dose Modifications. All patients with 
grade 4 non-hematologic toxicity were to be 
removed from protocol. If signs of cardiac 
toxicity developed, the ejection fraction (EF) 
by MUGA or echocardiography would have 
been re-tested and, if the EF fell by >10%, 
the patient would have been removed. If 
grade-3 toxicity persisted, treatment would 
have been held until toxicity resolved to grade 0 or 1 and the same dose of armed-ATC would have 
been washed to eliminate DMSO. If washing did not reduce toxicity, then the treatment would have 
been resumed with a 50% reduction in the armed-ATC dose. If the toxicities continued, the IL-2 would 
have been stopped and the aATC infusions would have been continued at the reduced dose. If grade 3 
toxicity persisted, the aATC infusions would have been stopped. One patient at dose level 3 had her 
infusions stopped due to grade 4 toxicities (hypertension and headache associated with a subdural 
hematoma). Of the remaining 18 patients, none had their doses reduced due to persistent grade 3 
toxicities. One patient had a fatal congestive heart failure that was likely due to Digitoxin toxicity. Three 
additional patients were added to the dose level without a DLT.  
 

V05/12/15 
15 
2.25 Clinical Responses to Her2Bi-armed 
ATC. Although clinical responses are not typically a primary endpoint for phase I clinical studies, we 
evaluated responses to therapy based upon tumor measurements, in those patients with measurable 
disease, as well as impact of treatments on levels of tumor markers in those patients who were 
clinically positive for tumor markers prior to initiation of treatments.  One partial remission was obtained 
and more than half of the patients remain stable with no evidence of increase in tumor size or 
development of new lesions at their follow-up, 
one month after completion of the last 
Her2Bi-armed ATC infusion.  Percentages of 
patients who remained stable during and after 
treatment with Her2Bi- armed ATC appear to 
increase as a function of treatment dose level 
(Tables 5 and 6). Serum tumor markers 
exhibited 
impressive 
decreases 
in 
Carcinoembryonic Antigen (CEA), CA 27.29, 
and Her2 receptors in the serum within one 
month after infusion Her2Bi-armed ATC (Table 
7).  
 
2.26 Overall Survival. For these analyses, we 
evaluated patients with tumors that had been 
identified as having HER2(3+) expression 
levels 
to 
patients 
having 
HER2(0-2+) 
expression levels (Fig-11). One patient with an 
unknown HER2 status was analyzed with the 
Her2 0-2+ group.  The median OS is 56 months 
for the Her2 3+ group, is 20 months for the 
Her2 0-2+ group, and is 26 months for the 
entire group with median follow-ups of 27.57 
(10.8-68.7), 16.54 (0.93-54.2), and 21.3(0.93-
68.7) months, respectively. 
 
2.27  Documentation of Partial Response in Her2 Negative Patient.   
A partial responder in the Her-negative group had 2 well-defined liver metastases on a PET/CT (2.5 x 
1.7 cm and 2.5 x 1.3 cm as shown in Before, Fig-12).  Reimaging after IT showed regression of the 
two lesions after 6 months (After Fig-12). The two lesions in the section disappeared. There was one 
small lesion that remained in a posterior section (not shown). The sum of longest diameters (LDs) was 
decreased by 30% at 4 weeks after IT and was decreased by >70% at 6 months. 
 
Fig-11 

V05/12/15 
16 
Fig-13 
 
2.28 Immune 
Responses as a Function of Her2Bi-armed 
ATC infusions. PBMC were obtained just prior to 
the indicated infusion number (Inf #) to evaluate 
steady-state (no ex vivo stimulation) IFNγ EliSpots 
(3-4 days after the last armed ATC infusion) and 
stimulated (SK-BR-3) IFNγ EliSpots. Four patients 
(1 with HER2 [3+] and 3 with HER2[0-2+]  were 
tested. Three of 4 of the patients had significant 
increases in specific cytotoxicity after Inf #4 
(without stimulation) that persisted after treatment 
(Fig-13).  Stimulation of the PBMC with SK-BR-3 
did not increase the Elispots in the pre-Inf #8 
samples. Fig-14 shows a representative patient in 
whom the IFNγ EliSpots and serum IFNγ levels 
represent ongoing pre-existing in vivo activation of CTL 
activity. The IFNγ EliSpots and serum IFNγ levels 
peaked prior to Inf #5 suggesting that 3-4 infusions was 
sufficient to maximally activated the endogenous 
immune system. These data suggest that IFNγ EliSpots 
will be sufficient to provide a surrogate marker for 
specific cytotoxicity directed at SK-BR-3 targets.  All 
together, IFNγ EliSpots and specific cytotoxicity appear 
to correlate even with small numbers of samples. The 
need for specific monitoring serum IFNγ would not be 
warranted. 
 
2.29 Enhanced Specific Cytotoxicity in Patient 
PBMC after Infusions of Her2Bi-armed ATC as 
Function of Time after Infusions. Five patients 
undergoing 
Her2Bi-armed 
ATC 
infusions 
were 
studied for cytotoxicity directed at SK-BR-3 before, 
during, and after treatment.  Fig-15 shows 3 of 5 and 
the Fig-16 shows 2 of the 5. The PBMC from the 2 
patients in Fig-16 had their PBMC separated into 
IgG2a+ and IgG2a- subpopulations to determine 
which populations mediated specific cytotoxicity. A 
significant increase in specific cytotoxicity directed at 
SK-BR-3 cells by PBMC obtained from all patients 
was observed during the course of treatment, with 
peak levels ranging from 14.15 ± 3.08% to 30.71 ± 
0.54%.  Specific cytotoxicity tended to increase during 
treatment with peak cytotoxicity occurring around 
infusion #8120. Cytotoxicity persisted up to 4 months 
after immunotherapy in patient FG1764. These data 
suggest that armed ATC not only persist but function 
in vivo.  
 
 
Fig-14:  Representative patient had PBMC tested for IFNγ 
EliSpots (left axis) and serum IFNγ (right axis) tested at the 
times indicated.  Fresh PBMC were plated in Elispot wells 
and the number of EliSpots were quantitated.     
 
Fig-12 

V05/12/15 
17 
2.30 Evidence for Activation 
and 
Immunization 
of 
Endogenous Immune Cells. 
When 
IFNγ 
EliSpots 
were 
performed on IgG2a-depleted 
blood from patients during and 
after immunotherapy, there were 
high 
numbers 
of 
as 
IFNγ 
EliSpots in the IgG2a negative 
population 
suggesting 
that 
endogenous 
cells 
were 
producing 
the 
immune 
response.  To confirm this 
observation 
using 
specific 
cytotoxicity, we tested IgG2a- 
populations 
from 
2 
patients 
(FG91801 and FG1792) for 
specific cytotoxicity directed at 
SK-BR-3 
targets120. 
Total 
PBMC obtained at times just 
prior 
to 
indicated 
infusion 
(Inf#) of armed ATC, original 
uncultured 
leukapheresis 
product, endogenous IgG2a- 
populations were prepared by 
depleting IgG2a+ cells from 
the PBMC sample. The IgG2a- 
depleted PBMC were tested in 
specific 
cytotoxicity 
against 
SK-BR-3 or Raji target cells 
(Fig-16). 
Taken 
together, 
these data together with the 
IFNγ-EliSpot 
data 
(section 
3.18, Fig-13) provide very 
strong 
complementary 
arguments that endogenous 
immune cell populations are involved in the 
specific 
cytotoxicity 
observed 
after 
infusions.  
 
2.31 Cytotoxicity Mediated by PBMC of 
Women Treated with Her2Bi armed ATC 
1 Week after Therapy.  Specific cytotoxicity 
of PBMC from 8 patients directed at SK-BR-
3 and Raji (irrelevant control) was tested 1 
week after completing immunotherapy using 
at E/T of 10:1 (Fig-17). Her2Bi-armed ATC 
treatments 
significantly 
increased 
cytotoxicity over cryopreserved PBMC from 
the leukapheresis product (p = 0.02). The 
solid black circles are Her2 positive patients 
and the open circles are Her2 negative 
patients. These data show that there was a 
statistically significant increase in the 
Fig-16: Pheresis, Armed ATC, unarmed ATC and IgG2a- Populations Were Tested for 
Specific Cytotoxicity Directed at SK-BR-3.  Specific cytotoxicity (E/T = 10:1) was 
performed in 51Cr assays.  IgG2a+ were depleted using Miltenyl beads.  The clear bars 
show cytotoxicity directed at SK-BR-3 targets and the solid bars show cytotoxicity 
directed at Raji targets. 
Fig-15:  Specific Cytotoxicity Mediated by PBMC obtained at the Designated Time 
Points from Patients Directed at SK-BR-3 (open bars) and Raji (solid bars). 51Cr was 
performed at an E/T of 10:1. PBMC were obtained from BrCa patients at the time of 
leukapheresis to obtain leukocytes for ATC expansion, prior to the 5th, 6th, and 8th 
infusions, 1 wk, 1 mo, 2 mo, and 4 mo after armed ATC infusions.  
Fig-17:  Specific Cytotoxicity by Patient PBMC Directed at SK-BR3 
Increases While Nonspecific Killing of Raji Targets Remains Low.  51Cr 
release was performed with 40,000 adherent targets at a E/T ratio of 10:1. 
Two Her2 0+ negative patients are indicated by grey and open circles. The 
solid black circles indicate Her2 3+ positive patients. 

V05/12/15 
18 
specific cytotoxicity mediated by PBMC from the women who 
received immunotherapy while non-specific cytotoxicity directed 
at the Raji lymphoma cell line remained low.  Both Her2 
negative patients also showed an increase in their specific 
cytotoxicity directed at SK-BR-3 targets.  These data suggest 
that the Her2 negative patients can also be induce by armed 
ATC infusions to develop specific cytotoxicity. 
  
2.32 Sequential Serum Cytokine Th1/Th2 and Type 1/Type 
2 Ratios in Patients. Serum samples, taken at indicated time 
points over the course of armed ATC infusions, were tested for 
cytokines the indicated cytokines using the Bio-Plex System. 
Fig-18 shows the mean Th1/Th2 and Type 1/Type 2 ratios for 7 
patients.   Both ratios shifted and remained polarized toward 
Th1 or Type 1 response throughout treatment.  The overall 
immune response, calculated as the average ratio of remained 
polarized towards a Th1–type response throughout treatment 
though more strongly so at the beginning of the infusions (9.46 
at Inf #1) than at the end (1.42 at Inf #8). These observations 
show a shift toward anti-tumor activity as a function of armed 
ATC infusions. 
 
2.33 Increased Levels of Serum IL-12 in Patients Receiving 
Her2Bi-armed ATC Infusions. Since IL-12 is produced mainly 
by activated monocyte/macrophages and IL-12 is the principal 
cytokine for polarizing T cell responses towards a Th1 
phenotype. 
We 
tested the serum of the patients for IL-12.  IL-12 is known to 
enhance the cytotoxic functions of NK and CD8+ T cells.  
Serum levels of IL-12 increased around 2 weeks or after 4 
infusions (Fig-19). These data are critical in that they 
suggest 1) armed ATC infusions establish a systemic Th1-
type anti-tumor milieu and 2) endogenous cells of the 
monocyte/macrophage lineage are activated to produce IL-
12 under conditions elicited by armed ATC infusions. The 
high serum levels of IL-12 show that the endogenous 
immunity has been activated in a robust manner, making a 
major contribution to the Th1 response observed in Fig-18.  
Armed ATC were not the source of the IL-12 since T cells 
do not produce IL-12. 
 
2.34 Induction of Serum IL-2 and GM-CSF Levels after 2 
Weeks or 4 Infusions of Armed ATC. The serum 
concentration of IL-2 and GM-CSF were determined for 
several reasons. First, we asked whether we could detect 
the IL-2 and GM-CSF that was being given to patients.  Second, we wanted to assess the levels of IL-2 
and GM-CSF that would be induced by multiple infusions of armed ATC.  The mean levels of IL-2 
(upper panels) and GM-CSF (lower panels) markedly increased by the 4 for 5th infusion (Fig-20).  Low 
levels of IL-2 (<100 pg/ml) were detected until the 5th infusion and low levels of GM-CSF were detected 
up until the 4th infusion.  The left panels show that subset of Her2/neu 0-2+ patients have similar 
response pattern to the entire group of patients (0-3+ group in the right panels.  The IL-2 data show that 
there is no increase or accumulation of the injected IL-2 up until the 3rd infusion (nearly 2 weeks of IL-2 
injections).  Similarly, there is no increase in serum GM-CSF until after the 3rd infusion (2 weeks after 
the twice weekly GM-CSF shots began).  Such low doses of IL-2 and GM-CSF given on the same 
 
Fig-19: Serum IL-12 Levels in 9 Patients. The 
mean is indicated by the horizontal bars. IL-12 was 
quantitated with the BioPlex. Serial samples were 
obtained prior to each infusion. 
Fig-18: Mean Ratio of Th1/Th2 (upper panel) 
and Mean Ratio of Type 1/Type 2 (lower 
panel). Th1/Th2 = [IL-2+IFN]/[IL-4+IL-5] and 
Type 1/Type 2 = [IL-2+IFN]/ [IL-4+IL-5+ 
IL-10+IL-13].  

V05/12/15 
19 
schedule 
after 
high 
dose 
chemotherapy 
and 
stem 
cell 
transplant 
for 
non-Hodgkin’s 
lymphomas could not be detected 
during the first 3 weeks after stem cell 
transplant 
(Lum, 
unpublished). 
Together these data suggest that 
infusions of IL-2  
and GM-CSF may help prime the 
immune system to induce a brisk Th1 
immune response.         
 
2.35 Her2Bi Persists on the ATC 
and Mediates Multiple Cycles of 
Specific Cytotoxicity. In studies 
evaluating the long-term activity and 
fate of Her2Bi-armed ATC, expansion 
and division of Her2Bi-armed ATC vs. 
unarmed ATC have been determined 
by comparing survival and ability of 
cells to divide and kill target cells 
when repeatedly exposed in vitro to 
SK-BR-3 cells over 336 hours of 
culture. Up to 2 weeks after a single 
arming, ex vivo expanded Her2Bi-armed ATC co-cultured with SK-BR-3 targets increase in number, 
undergo multiple cell divisions, mediate multiple rounds of specific cytotoxicity, and secrete both 
cytokines and chemokines without undergoing Fas/FasL-induced apoptosis or activation-induced cell 
death120.  In vitro, armed ATC divided, maintained surface Her2Bi (Fig-21), and expressed a range of 
activities for extended periods of time. Perforin-
mediated cytotoxic activity by armed ATC 
continued for at least 336 hours, and cytokines 
and chemokines (IFN-γ, TNFά, GM-CSF, MIPά, 
and RANTES) were secreted during successive 
rounds of SK-BR-3 stimulation120. Armed ATC 
would 
divide, 
kill, 
and 
secrete 
cytokines/ 
chemokines multiple times as a result of multiple 
rounds of targeting SK-BR-3 cells as a function of 
surface bound Her2Bi was detected by goat anti-
IgG2a (OKT3). Cytotoxicity persisted beyond the 
lower limits of detection for Her2Bi on the cell 
surface (Fig-21, right axis). Specific cytotoxicity 
by armed ATC and persistence of Her2Bi is 
shown in Fig-21.  Mean percent specific 
cytotoxicity (Left axis) directed at SK-BR3 
(E:T=10:1) for armed ATC (n=4) and the percent 
positive IgG2a+ cells at each time point by flow 
cytometry using polyclonal goat anti-mouse 
IgG2a PE-conjugated Mab to detect the OKT3 
(Right axis).  
 
2.36 Engagement of Her2Bi-Armed ATC 
Induces In Vitro Cell Cycling.  Her2Bi armed 
ATC were labeled 
 with CSFE-dye as a marker for cell proliferation and stimulated with SK-BR-3 to determine whether 
Fig-20: Serum IL-2 and GM-CSF Levels. The mean is indicated ± S.D. Serial 
serum samples collected at the indicated time points were quantitated with the 
BioPlex.  
Fig-21: Shows % Specific Cytotoxicity (Left Axis) and % 
Her2Bi armed ATC (IgG2a+ cells) by Goat anti-mouse IgG2a 
flow cytometry. 51Cr release was assessed at an E/T of 25:1.  
Armed ATC were repeated exposed to SK-BR3 targets at 0, 
48, 96, 213, and 336 hours after a single arming with Her2Bi.  
After each exposure, the armed ATC were recounted after the 
time interval, washed, adjusted, and replated onto a fresh set 
of SK-BR-3 targets.  The means of 4 experiments is 
presented.  Unarmed ATC tested in parallel did not expand or 
continue to kill at high levels.   

V05/12/15 
20 
engaging the ATC via their Her2Bi receptor would induce proliferation. We found that armed ATC 
would divide up to 4 times in a 48 hrs (Fig-22A) whereas unarmed ATC (Fig-22C) underwent far fewer 
cell divisions after 48 hrs (Fig-22D).  These data, together with the cytotoxicity data in section 3.25, the 
persistence of IgG2a on the surface of ATC, and cytokine secretion in vitro and in vivo suggest that 
infused armed ATC persist, induce an immune response that “vaccinates” the patient to his own “tumor 
antigens”, may proliferate in vivo with repeated stimulation with tumor antigens, and induce or recruit a 
systemic immune response to leads to high levels of circulating cytokines in the serum of patients.  
 
2.37 Phase I Clinical Trial in High Risk Breast Cancer (≥ 4 nodes).  In order to test whether Her2Bi 
armed ATC would affect time to progression, nine women received 8 doses of Her2Bi armed ATC twice 
per week for 4 weeks in combination with daily IL-2 (300,000 IU/m2/day and twice weekly GM-CSF 
(250 µg/m2/twice weekly) beginning 3 days before the first 
infusion and ending 7 days after the last infusion.  There 
were no dose limiting toxicities.  Five of 9 patients are 
alive free of disease with a median overall survival of 103 
months (Fig 23). 
 
2.38 Human Anti-Mouse Antibody (HAMA) responses.  
Because one mAb moiety comprising Her2Bi is an 
unmodified mouse mAb, we evaluated patients’ sera for 
the development of HAMA. Of the 11 patients evaluated, 
none developed clinically significant HAMA levels (>10 
ng/ml) during or following therapy. Average HAMA 
concentrations in patients did not differ significantly as 
function of dose level (p =0.55).  There was no correlation between levels of IgG2a+ cells in patients’ 
peripheral blood (Fig-24) and their respective HAMA levels (r2 = 0.001; p = 0.86). Therefore, HAMA 
responses to the IgG2a (OKT3) component of Her2Bi do not appear to play a role in the clearance of 
armed ATC.   
 
2.39 Summary of Preliminary Data: Our in vitro and in vivo clinical data show that armed ATC: 1) 
infusions are feasible and safe; 2) have well demonstrated in vitro activity against MCF-7 and Sum 
1315 cell lines that have very low and nearly absent Her2 expression; 3) cryopreserved patient 
products mediate levels of specific cytotoxicity comparable cytotoxicity exhibited prior to 
cryopreservation; 4) sensitive flow cytometry methodology 
was developed to detect and follow the kinetics and 
persistence of armed ATC in the blood of patients; 5) 
111Indium labeled armed ATC provide information on the 
trafficking of armed ATC up to 4 days after infusions; 5) 
overlay double staining of tumor biopsies detect the 
presence of armed ATC in tumors 1 week to 1 month after 
therapy; 6) serum tumor markers exhibited impressive 
decreases in CEA, CA27.29, and Her2 receptors; 7) 
armed ATC infusions may have improved OS for the both 
Her2 0-2+ (median=20 months) and Her2 3+ patients 
(median=56 
months); 
8) 
infusions 
induced 
the 
development of BrCa specific CTL activity that parallel 
IFNγ EliSpots data that argue for a robust adaptive 
endogenous response with a statistically significant 
increase in the CTL activity following immunotherapy; 9) 
infusions polarized the immune system to a Th1/Type 1 
that was associated with a remarkable increase in IL-12 
production that occurred within 2 weeks after initiation of 
immunotherapy; 10) injections of IL-2 and GM-CSF did 
not increase serum levels of IL-2 and GM-CSF during the 
Fig-24:  HAMA Responses to IgG2a Were Measured 
by  ELISA. Human anti-mouse antibodies (HAMA) by 
ELISA (Antigenix, Inc. Huntington Station, NY). 
Individual patient samples are shown as solid circles 
and horizontal bars represent the mean +/- SEM for 
the number of patients sampled at each time point (n). 
0
20
40
60
80
100
120
140
0
20
40
60
80
100
>10 nodes
Median = 103.5 months), n = 9
Adjuvant Breast Cancer (Her2 0-3+)
for High Risk (>10 +nodes)
Months
Percent survival
Fig-23 

V05/12/15 
21 
first 2 weeks of therapy (showing that levels did not accumulate) and there was a remarkable rise in all 
of the Th1 cytokines after 2 weeks with levels that could not be due to IL-2 and GM-CSF injections 
(demonstrating that endogeneous activation was responsible for the rising levels); 11) the preclinical 
data showing the capacity of armed ATC to repeatedly kill, proliferate, and secrete 
cytokines/chemokines combined with the flow cytometry data that show circulating armed ATC and 
armed ATC in biopsy specimens suggest that the armed ATC persist and survive long enough to 
vaccinate the patient against their own tumor antigens; and 12) immunotherapy consisting of aATC, IL-
2, and GM-CSF provides strong evidence for anti-breast cancer effect in phase I high risk ≥ 4 nodes 
breast cancer patients who received adjuvant therapy with median OS of 103.5 months with 5 of 9 
women alive without disease. Together these data provide compelling evidence for the proposed 
clinical trial strategy and immune evaluation function tests to identify in vivo/in vitro correlates.  
 
3.0 
ELIGIBILITY 
3.1 Inclusion Criteria:  
Patients who satisfy all of the following conditions will be considered eligible for the study: 
3.1.1 Signed and dated IRB-approved consent form  
 
3.1.2 Patients may be male or female. 
 
3.1.3 18 years of age or older. Women of reproductive potential must agree to use an effective 
non-hormonal method of contraception during therapy. 
 
3.1.4 ECOG performance status (PS) of 0 or 1 and/or Karnofsky PS of ≥70%. 
 
3.1.5 Diagnosis of invasive adenocarcinoma of the breast made by core needle biopsy. 
 
3.1.6 Palpable primary breast tumor measuring ≥ 2.0 cm on physical exam or imaging prior to 
neoadjuvant chemotherapy. 
 
3.1.7 Patients with stage II-III breast cancer that is HER2-negative by IHC (0-2+) or FISH 
(HER2/CEP17 amplification ratio <2.0) who have completed “third generation” neoadjuvant 
chemoT (see section 4.3.1) and planned local treatment (surgery and Radiation if 
indicated).  ER or PR receptors status can be ER negative (≤10% by IHC) or PR negative 
(≤10% by IHC). 
 
3.1.8 Patients may have lymph node positive or negative disease, as long as they have clinical or 
pathologic stage II or III breast cancer.  Patients may have the lymph nodes assessed by 
any method deemed appropriate by the treating physicians, including pre-neoadjuvant 
therapy sentinel lymph node biopsy.  
 
3.1.9 Presence of any residual disease on final pathology following surgery will be required for 
immunotherapy. Patients with no residual disease at the time of surgery will be removed. 
 
 
3.1.10 At the time of registration: 
 Absolute neutrophil count (ANC) must be ≥ 1000/mm3. 
 Platelet count must be ≥ 100,000/mm
3. 
 Hemoglobin must be ≥ 9.0 mg/dL. 
 There must be evidence of adequate hepatic function by these criteria: 
− Total bilirubin must be ≤ the ULN for the lab unless the patient has a grade 1 bilirubin 
elevation (> ULN to 1.5 x ULN) resulting from Gilbert’s disease or similar syndrome 
due to slow conjugation of bilirubin; and 
− Alkaline phosphatase must be ≤ 2.5 x ULN for the lab  
− AST/ALT must be ≤ 1.5 x ULN for the lab. 

V05/12/15 
22 
− Alkaline phosphatase and AST/ALT may not both be > the ULN. For example, if the 
alkaline phosphatase is > the ULN but ≤ 2.5 x ULN, then the AST/ALT must be ≤ the 
ULN. If the AST/ALT is > the ULN but ≤ 1.5 x ULN, then the alkaline phosphatase 
must be ≤ ULN. 
 
3.1.11 Patients with either skeletal pain or alkaline phosphatase that is > ULN must have a bone 
scan showing they do not have metastatic disease. Suspicious findings on bone scan 
must be confirmed as benign by x-ray, MRI, or biopsy. 
 
3.1.12 Patients with AST/ALT or alkaline phosphatase > ULN must have liver imaging that does 
not demonstrate metastatic disease. 
 
3.1.13 Patients with AST/ALT >ULN must have negative hepatitis studies. 
 
3.1.14 Patients with stage II disease and clinical suspicion for metastatic disease based on 
reported symptoms, physical examination findings, or laboratory abnormalities must have 
staging studies demonstrating no evidence of metastatic disease (with exception of 
axillary lymph nodes or mammary  nodes).  Patients with stage III disease must have 
staging studies demonstrating no evidence of metastatic disease (with exception of 
axillary lymph nodes or mammary nodes), even if asymptomatic with normal physical 
examination and laboratory values.  Such staging studies must include: chest imaging 
(chest X-ray, CT, or MRI), abdominal/pelvis imaging (CT or MRI), and bone imaging (bone 
scan or PET-scan).  Abnormalities that are indeterminate and too small to biopsy should 
be followed with further imaging, as appropriate, but do not exclude patients from the 
study.  Abnormalities that are suspicious and large enough to biopsy exclude patients 
from the study, unless a biopsy is performed and is negative for metastatic disease. 
 
3.1.15 Serum creatinine ≤ 1.5 x ULN for the lab. 
 
3.1.16 Left Ventricular Ejection Fraction (LVEF) ≥ 45 % (by MUGA or echocardiography) 
 
3.2  Exclusion Criteria: 
Patients with any of the following conditions will be ineligible for this study: 
3.2.1 Tumor determined to be HER2-positive by immunohistochemistry (3+) or by fluorescent in 
situ hybridization (HER2/CEP17 amplification ratio ≥2.0). Tumors determined to be ER or 
PR positive by immunohistochemistry (>10% ). 
 
3.2.2  Tumors clinically staged as unresectable disease.  
 
3.2.3 Evidence of disease progression on Neoadjuvant chemo T 
 
3.2.4  Definitive evidence of metastatic disease with exception of axillary lymph nodes or 
mammary nodes. 
 
3.2.5  Synchronous bilateral breast cancer (invasive or DCIS). 
 
3.2.6 Treatment with biotherapy, and/or hormonal therapy for the currently diagnosed breast 
cancer prior to study entry. 
 
3.2.7  Prior history of invasive breast cancer (Patients with a history of DCIS or LCIS are 
eligible.) 
 
3.2.8 Other malignancies unless the patient is considered to be disease-free for 5 or more years 
prior to randomization and is deemed by the physician to be at low risk for recurrence. 

V05/12/15 
23 
Patients with the following cancers are eligible if diagnosed and treated within the past 5 
years: carcinoma in situ of the cervix, carcinoma in situ of the colon, melanoma in situ, 
and basal cell or squamous cell carcinoma of the skin. 
 
3.2.9  Known cardiac disease which precludes their ability to receive planned treatments: 
 angina pectoris that requires the use of anti-anginal medication 
 history of documented congestive heart failure 
 serious cardiac arrhythmia requiring medication 
 severe conduction abnormality 
 valvular disease with documented cardiac function compromise; and 
 uncontrolled hypertension defined as BP that is consistently > 150/90 on 
antihypertensive therapy at the time of registration. (Patients with hypertension that is 
well controlled on medication are eligible.) 
 
3.2.10 History of myocardial infarction (MI) documented by elevated cardiac enzymes with 
persistent regional wall motion abnormality on assessment of LV function.  (Patients with 
history of MI must have an echo instead of/in addition to a MUGA to evaluate LV wall 
motion.) 
 
 
3.2.11  Other non-malignant systemic disease (cardiovascular, renal, hepatic, etc.) that would 
preclude treatment with any of the treatment regimens or would prevent required follow-up. 
 
3.2.12 Chronic ongoing oral steroid use at the time of registration for any condition (such as 
asthma, rheumatoid arthritis, etc). 
 
3.2.13 Administration of any investigational agents within 30 days before study entry. 
 
3.2.14 Pregnancy or lactation at the time of registration. 
 
3.2.15 Psychiatric or addictive disorders or other conditions that in the opinion of the investigators 
would preclude the patient from complying with the study protocol. 
 
Minor changes from these guidelines will be allowed at the discretion of the research team 
under special circumstances. The reasons for exceptions will be documented. 
 
3.3 Informed consent.  Each patient must be aware of the nature of his/her disease and must willingly 
consent to treatment after being informed of alternatives, potential benefits, side effects, and risks. 
 
3.4 Registration.  Patients may be enrolled after diagnosis if they have documented TNBC while they 
are undergoing neoadjuvant chemotherapy, surgery, and/or irradiation any time up to 3 months after 
completion of breast cancer treatment. During this time when they are undergoing standard of care 
treatment, they will undergo baseline studies,  any staging and eligibility testing as described in section 
3 prior to leukapheresis. Patients will be registered in the clinical trials office (CTO) of the Karmanos 
Cancer Institute (KCI) by the approved study coordinators.  The study coordinators may be contacted 
at (313)576-8384 or (313)576-8506 or (313)576-9277. 
 
3.5 Screening Assessments. Medical history, physical examination, EKG, CXR, breast imaging 
(mammography, ultrasound, or MRI) for tumor measurements, bone scans or PET-scans (if required), 
MUGA or echocardiography, laboratory tests (hematology, chemistry, urinalysis, creatinine clearance), 
and staging CT or MRI (if required) will be performed prior to registration as per section 3.1.14.  
 
4.0  TREATMENT PLAN 

V05/12/15 
24 
4.1 Overall Treatment Plan.  Patients with ER/PR negative and HER2-negative, stage II-III operable 
breast cancer with residual disease following completion of Neoadjuvant chemoT and local treatment 
will be entered into a phase II trial to evaluate whether infusions of HER2Bi-armed activated T cells 
after neoadjuvant chemoT improve PFS (primary endpoint).  
 
 
 
 
Neoadjuvant chemoT regimens using anthracycline and/or taxane-based, “third generation” 
chemoT  regimens (dose dense ACT or TAC), which are considered equivalent by current 
standards, will be used. (See section 4.3.)Dose and schedule modifications will be per treating 
physician’s discretion. 
 
After planned breast cancer treatment, PBMC will be collected during 1-2 leukapheresis 
sessions (utilizing a central venous access catheter), activated with anti-CD3 and expanded in 
IL-2 to generate ATC that will be armed with HER2Bi, washed, and cryopreserved in 8 aliquots 
for subsequent infusions.  
 
Armed ATC will be thawed and patients will receive 8 infusions of ~10-15 billion armed 
ATC given twice per week for 4 weeks for a total dose of ~80 billion for each patient. The first 
infusions of armed ATC will be given no sooner than 3 weeks (±1 week) after completion of 
breast cancer treatment. 
 
Immune function tests will be performed before, during and after IT at the designated time 
points.  
 
Tumor samples will be obtained at diagnosis if possible (i.e. archived tissue from the diagnostic 
biopsy performed prior to study registration per routine care) and at the time of surgery. Tumors 
samples will be tested for CSC phenotyping and subsets of infiltrative cells. 
 
See the study calendar (section 8) for additional information. 
 
4.2 Activation and Expansion of T cells with OKT3 and IL-2.  Armed ATC are prepared in the 
Cancer Immunotherapy Laboratory’s cGMP facility (see Section 7).  Immediately after leukapheresis, 
the lymphocytes are activated with soluble monoclonal anti-CD3 antibody (OKT3), which cross-links the 
CD3 receptors on T cells and activates them.  The ATC are expanded in the presence of IL-2 for up to 
14 days.  After culture, ATC are harvested, armed with OKT3 x Herceptin® (Her2Bi), washed to remove 

V05/12/15 
25 
unbound Her2Bi,  and cryopreserved in 10% DMSO and 20% protein (human albumin or serum) using 
rate controlled freezing and storage in liquid nitrogen.  No exogenous IL-2, OKT3, or other culture 
reagents (e.g. medium components) are present in the final cryopreserved product.  Armed product is 
released for clinical use after Quality Control testing for sterility (bacterial and fungal culture, endotoxin 
and mycoplasma), phenotype (% of CD3 cells), and activity (i.e. cytolytic activity against the SKBR-3 
cell line).  
 
4.3 Neoadjuvant Chemotherapy.   
4.3.1 Neoadjuvant chemoT may be administered at any location where the treating (KCI or non-
KCI) oncologist normally administers chemoT.  Acceptable standard, “third generation” 
regimens are recommended. 
 
4.3.2 Every effort should be made to administer full doses of the standard therapy to ensure the 
best efficacy; however, dose modifications may be made, if necessary, as determined by the 
treating physician.  Every effort should be made to administer the chemotherapy on the 
schedule to ensure the best efficacy; however, dose delays may be necessary for significant 
toxicity, as determined by the treating physician. 
 
4.3.3 Treating physicians should perform regular histories/physical examinations and monitor 
appropriate laboratory studies during chemotherapy per good medical practice.   
 
4.3.4 Tumor assessments will be performed by the treating physician as clinically appropriate 
during and after the neoadjuvant chemoT to document response to therapy. 
 
4.3.5 Patients who discontinue any part of the neoadjuvant chemoT due to unacceptable toxicity 
will remain eligible to receive the aATC treatment and proceed to this followed by curative 
surgical resection as planned. 
 
4.4  Immunotherapy with Armed ATC, IL-2, and GM-CSF 
 
4.4.1 HER2Bi armed ATC Infusions   
4.4.1.1 Cardiac evaluation: As a MUGA or echocardiogram is due to be repeated following 
chemotherapy, the LVEF must remain ≥ 45% in order to proceed with aATC infusion.  
If the LVEF drops below 45%, the patient will be ineligible for the study. 
 
4.4.1.2 Armed ATC infusions will be done in the Immunotherapy outpatient clinic. All 
appropriate assurances for identification of product, patient, sterility, etc, will be 
performed prior to infusion according to the SOPs for the armed ATC IND.  Frozen 
armed ATC will be thawed at the bedside of the patient just prior to infusion. If there 
is evidence of infusion-related toxicities, subsequent armed ATC will be thawed, 
washed, and resuspended in medium prior to infusion. The first armed ATC infusion 
will begin 3 weeks (±1 week)  after all breast cancer treatment and subsequent 
doses will be administered twice weekly for a total of 8 doses.  Armed ATC will be 
infused intravenously (IV) at a rate based on the endotoxin content of the product 
through a central venous access catheter (to minimize the risk of clotting)..  All 
patients will be observed for at least 4 hours after each infusion. If stable, patients 
will be discharged home. 
 
4.4.1.3 If clinically indicated, additional evaluation studies (one 10 ml red top tube) may be 
drawn at any or all of the following time points:  pre-infusion, 1, 2, and 4 hours (+/- 15 
min), 8 and 16 hours (+/- 1 hr), 24, 48, and/or 72 hours (+/- 2 hr) after each infusion, to 
study cytokine release.  “Clinically indicated” is based on the clinical judgment of the 
investigator. 
 
 
 

V05/12/15 
26 
4.4.2 Recommended Concomitant Medications:  All patients will be pre-medicated with 
Benadryl (50 mg IV or PO) 30 minutes prior to each Her2Bi-armed ATC infusion.  This medication 
may be repeated every 4-6 hrs as needed. Demerol (25-50 mg IV) will also be administered if the 
patient develops grade 3 chills and then may be administered 30 minutes prior to subsequent 
armed ATC infusions dependent upon the severity of the initial reaction.  Hydrocortisone (50-100 
mg IV) for severe adverse reactions related to armed ATC infusions may be used, if necessary.  
Patients will then be treated with hydrocortisone (50-100 mg IV) at each subsequent infusion 30 
minutes prior to infusion to prevent the reaction For grade 4 infusion reactions, patients will be 
removed from study treatment (aATC infusions), but patients may continue to participate in the 
remaining aspects of the study (tissue samples, immune studies, follow-up, etc) and will be 
included in the intent to treat analysis. (See section 5.4 for required dose modifications.) 
 
4.4.3  Subcutaneous IL-2 Injections:  All patients will receive SQ IL-2 (300,000 IU/m2/day), 
beginning 3 days before the first ATC infusion and ending 1 week after the last ATC infusion. 
 
4.4.4 GM-CSF Therapy: All patients will receive SQ GM-CSF (250 μg/m2/twice weekly), 
beginning 3 days before the first ATC infusion and ending 1 week after the last ATC infusion. 
 
4.4.5 Ancillary Therapy:  Patients will receive full supportive care including transfusion of blood 
and blood products, antibiotics, and anti-emetics, when appropriate.  The reason(s) for treatment, 
dosage and the dates of treatment will be recorded.  Patients will receive calcium replacement 
during the pheresis procedure, if needed for symptoms of hypocalcemia. 
 
4.4.6 Steroids/Other Therapy: With the exception of steroids for adrenal failure, septic shock, 
pulmonary toxicity or hormonal therapies administered for pre-existing, non-disease-related 
conditions (e.g. insulin for diabetes), steroids and hormonal therapies will not be administered.  
Hydrocortisone (50-100 mg IV) for severe adverse reactions related to armed ATC infusions is 
allowed. 
 
5.0 POTENTIAL TOXICITIES, DOSE MODIFICATIONS, AND MANAGEMENT 
5.1 Pheresis Procedure.  Reported toxicity from pheresis is minimal.  Patients have a small potential 
for infection from the placement of central catheters used for pheresis.  Use of the anticoagulant citrate 
dextrose may cause symptoms of mild hypocalcemia, which are controlled with calcium replacement 
during the procedure, as necessary. 
 
 
5.2 Her2Bi-armed Activated T Cells. Severity of toxicities associated with Her2Bi-armed ATC has 
been minimal (see Table 4) and symptoms have been responsive to interventional therapy with 
antihistamines and analgesics.  The most common side-effects are chills and headache.  Other 
reported side-effects included mild gastrointestinal (nausea) symptoms and backache.  Adverse 
symptoms associated with cryopreservative (i.e. DMSO) accounts for approximately 50% of all infusion-
related side-effects.  DMSO-related side effects include fever, nausea and fatigue. No fatalities have 
occurred as a result of receiving Her2Bi-armed ATC infusions.  
 
5.3 Dose Modification for HER2Bi Armed-ATC Toxicity.  All patients with treatment related, grade 4 
non-hematologic toxicity will be removed from protocol. If there is clinical evidence of heart failure, a 
MUGA or echocardiography will be done to evaluate the left ventricular ejection fraction (LVEF).  If it 
falls by >10% from the previous value (by MUGA or echocardiography) following HER2Bi armed-ATC, 
further therapy will be withheld and the patient will be removed from protocol treatment.  If there is 
persistent grade 3 toxicity at any time, treatment will be held until toxicity improves to grade 0 or 1.  For 
patients who experience grade 3 infusion reactions, the same dose of armed-ATC will be washed prior 
to all subsequent dose administrations to eliminate DMSO and protein.  If washing does not reduce 
toxicity, then the treatment will be resumed with a 50% reduction in the armed ATC for all subsequent 
doses in that course. If a grade 3 toxicity occurs again, the armed ATC infusions will be stopped.  
Toxicity will be assessed via telephone contact between each aATC infusion and up to 7 days beyond 

V05/12/15 
27 
the last aATC infusion.  Additionally, patients will be given contact numbers and will be instructed to call 
and report any urgent problems associated with the aATC infusions.  Patients for whom the aATC 
infusions are stopped will remain on study and complete the other study procedures (tissue samples, 
immune studies, etc). 
 
6.0 DRUG INFORMATION 
6.1 Commercial ChemoT Doxorubicin, paclitaxel, docetaxel, cyclophosphamide, and G-CSF must be 
obtained from commercial sources. Please refer to the current FDA-approved package inserts provided 
with the chemoT or the Physicians’ Desk Reference for information about possible side effects and 
instructions for preparation, handling, and storage of the drugs. 
   
 
6.2  OKT3. This is a murine IgG2a monoclonal antibody directed at human CD3 commercially 
available from Miltenyi Biotec, Auburn, CA.  It is purchased in vials containing 5 mg/5 ml of 
reconstituted bacteriostatic water.  OKT3 is used to activate T cells for growth and for heteroconjugation 
with Herceptin® to produce the Her2Bi bispecific antibody for arming patient ATC. 
 
 
6.3 Anti-HER2/neu monoclonal antibody (Herceptin®, Trastuzumab; Genentech, Inc., CA).  
Herceptin is a humanized murine monoclonal antibody directed at HER2/neu, and is commercially 
available in multi-dose vials containing 440 mg of drug.  For this study, Herceptin® is heteroconjugated 
to OKT3 to produce the Her2Bi bispecific antibody for arming patient ATC.  
 
6.4 Anti-CD3 (OKT3) x anti-HER2/neu heterconjugated bispecific monoclonal antibody (HER2Bi).  
Anti-CD3 x anti-HER2 is produced under GMP conditions. IND #9985 was cleared for clinical trials by 
the FDA that specifies the production of bispecific antibody, sterility testing, and the standard operation 
procedures for arming of activated T cells.  The Her2Bi has been retested for targeting and cytotoxicity 
and shown to be stable for over 3 years stored at 4oC. 
 
6.5 Interleukin 2 (IL-2).  
 
6.5.1 Therapeutic classification: lymphokine 
 
6.5.2  Known Side Effects and Toxicities: Administration of low dose SQ IL-2 (2  105 
IU/m2/day) for 90 days resulted in no grade III toxicities.  None of the patients experienced 
pulmonary capillary leak syndrome, severe hypotension, oliguria, azotemia, or hyperbilirubinemia.  
The most frequent toxicities included fatigue, fever, and nausea.  None of the patients had to stop 
their SQ IL-2 therapy due to side effects.  Therefore, it is unlikely that major toxicities associated 
with the low dose SQ IL-2 will occur although death due to high dose IL-2 toxicity has been 
reported 
 
6.6. Granulocyte-macrophage colony stimulating factor (GM-CSF):  
 
6.6.1  Therapeutic Classification: colony stimulating factor. 
 
6.6.2  Known Side Effects and Toxicities: Patients receiving GM-CSF (Leukine-Sagramostim) 
have experienced fever 60-90 min after administration (duration 1-4 hrs); chills; nausea; vomiting; 
diarrhea; fatigue; weakness; headache; decreased appetite; thrombosis; rapid or irregular 
heartbeat or other heart problems; feeling of faintness; facial flushing; pain in the bones, muscles, 
chest, abdomen, or joints; local reaction at the site of injection; rashes; and kidney and liver 
dysfunction.  Eosinophilia or other blood component abnormalities may occur.  There have been 
infrequent reports of fluid accumulation or worsening of preexisting fluid accumulation in the 
extremities, in the lungs, and around the heart which may result in breathing problems or heart 
failure.  Rarely, patients have developed acute allergic reactions.  There have also been reports 
of low blood pressure, hypoxia, transient loss of consciousness, and difficulty in breathing after 
the first injection of Sargramostim.  These signs may or may not recur with additional injections of 

V05/12/15 
28 
Sargramostim.  Patients with prior heart, lung, kidney, or liver problems may have worsening of 
their symptoms following administration of Sargramostim.  There may be other side effects that 
could occur. 
 
7.0  PREPARATION OF HER2Bi ARMED ACTIVATED T CELLS  
7.1 Facility.  Qualified personnel who are familiar with procedures which minimize undue 
exposure to themselves and to the environment will undertake the preparation, handling, and safe 
disposal of immunotherapeutic agents in a self-contained protective environment. Cells will be 
generated in the Cancer Immunotherapy Laboratory under FDA #BB-IND 9985 with standard 
operating procedures (SOPs) for growing, splitting, harvesting, arming of ATC, cryopreservation, 
and infusion of armed ATC. 
 
7.2 Activation, Culture, and Freezing of Armed-ATC.  Lymphocytes are obtained by 
leukapheresis and cultured at a density of 1-3 × 106 cells/ml in RPMI 1640 media (Lonza) 
containing 100 IU/ml of IL-2 (Novartis), 10-20 ng/ml of OKT3 (Centocor Ortho Biotech), and 2% 
human serum (Biowhitaker/Valley Biomedical).  Cells will be cultured for a maximum of 14 days in 
medium to which  no additional OKT3 will be added during the expansion period. 
 
7.3 Preparation of anti-CD3 x anti-HER2 (Her2Bi) Bispecific Antibody.  The specific details for 
the production, purification, and quality control testing are part of IND #9985. 
 
7.4 Arming of ATC with HER2Bi.  The harvested ATC will be counted and a dose of 50 ng of 
HER2Bi per million ATC will be added to the solution and incubated for 1 hr at 4oC.  The armed 
ATC will be washed, counted, and resuspended in the final solution that will be cryopreserved in 
aliquots specific for each infusion. 
 
7.6 Cytotoxicity Assay and IFNγ EliSpots.  Cytotoxicity is measured in a 20 hr 51Cr -release 
assay to ensure activity (minimum ≥ 10% cytotoxicity) of armed ATC over their unarmed 
counterparts.  Tumor target cells are plated in a flat-bottomed microtiter plate and incubated at 
37oC.  The targets are washed and labeled the next day with 51Cr at 37oC.  These wells containing 
tumor cells will be washed and armed or unarmed ATC will be added at different E:T ratios for 20 
hr incubation at 37oC.  The next day, the supernatants harvested from the microtiter wells will be 
counted and the percent specific lysis will be calculated.  IFNγ EliSpots will performed by 
plating SK-BR-3 targets. 
 
7.7 Quality Assurance of armed ATC Cell Product. Lists of suppliers of monoclonal antibodies, 
heteroconjugation reagents, and culture reagents will be maintained as well as lot numbers used 
and supplier-provided documentation of sterility and documentation that all reagents are free of 
endotoxin and mycoplasma. Records of all quality control measures will be maintained by the 
laboratory. 
 
8.0 REGISTRATION AND REQUIRED DATA 
8.1 
Pre-Study.  All patients enrolled will be evaluated prior to initiation of therapy.  Eligibility 
criteria will be assessed as stated in section 3.0. Patients may be enrolled at any time after 
diagnosis until 3 months after completion of their breast cancer treatment.   The patients will be 
leukapheresed after enrollment to obtain lymphocytes for expansion or when the lymphocyte 
population has recovered to ≥ 500 lymphocytes/mm3 

V05/12/15 
29 
8.2 
Study Calendar   
 
Pre-
Registration 
Pre and 
Mid IT1 
After IT  
History & Progress Notes 
X 
X 
2 weeks, 3 months, 6 months and 12 
months post IT2 
Physical Exam 
X 
X 
2 weeks, 3 months, 6 months and 12 
months post IT2  
Performance Status 
X 
X 
2 weeks, 3 months, 6 months and 12 
months post IT2  
Laboratory: 
--CBC/Plts/Diff 
X 
X 
2 weeks, 3 months, 6 months and 12 
months post IT2  
--Lytes, Bili, AST, ALT, BUN, Cr, CrCl 
X 
X 
2 weeks, 3 months and as clinically 
indicated2 
--Urinalysis 
X 
 
As clinically indicated 
--Serum Pregnancy 
X 
 
 
--EKG 
X 
 
As clinically indicated 
--MUGA or Echocardiography3 
X 
 
 As clinically indicated 
--HBsAg 
X 
 
As clinically indicated 
--CEA, CA 15-3, or CA 27.29  
As indicated 
 
As clinically indicated 
Staging: 
--Bone scan or PET-scan 
As indicated 
 
As clinically indicated 
--CXR, CT or PET/CTMRI of Chest; 
As indicated 
 
Pre treatment for staging and  clinically 
--Mammography/, ultrasound, or MRI 
 X 
 
As clinically indicated 
--Others (CT Head, etc) 
As indicated 
 
As clinically indicated 
 
 
 
 
 
 
 
 
Immune Evaluations6  
 
X6 
Just prior to 1st IT(within 3 days),after 4 
IT infusions and 2 weeks (±3 days) or 1 
month (±3 days) post starting 
immunotherapy; studies at 3, 6, and 12 
months after IT, if immune responses 
are positive and continue to be of 
interest to the investigators2. 
1    PreIT: (IT=armed ATC) the day of IT or up to 7 days prior.Mid IT:Preinfusion #5 
2   To be completed within the following timelines: 2 weeks (±3 days), 3 months (±1 week), 6 months 
(±1 months), and 12 months (±1 months) post IT. 
3    Ejection fraction by MUGA or echo should be >45% 
4    Sample obtained from the diagnostic biopsy if available and from surgical specimen 

V05/12/15 
30 
5  Immune studies will be done at leukapheresis,  preinfusion #5, and 2 weeks (±3 days) after the 
last armed ATC infusion or 1 month (±3 days) after the initiation of immunotherapy. Additional studies 
will be done at 3 months (±1 week), optional 6 months (±1 month), and optional 12 months (±1 
month) after the last infusion if studies are positive. If the immune studies show changes worthy of 
follow-up in the opinion of the investigators, optional studies can be obtained at 6 month (±1 month) 
intervals, until the results become negative. 
 
9.0 CRITERIA FOR EVALUATION (Cited criteria are based upon those established for the 
comparative study group)  
 
9.1 Tumor Measurements.  Tumor assessment (breast and axilla) will be measured on the final 
pathology from surgical specimen after completion of chemoT. Tumor measurements done 
clinically by physical exam and by mammogram and ultrasound in centimeters prior to neoadjuvant 
chemotherapy and prior to definitive surgery will be collected.  These measurements will consist of 
the longest diameter and the perpendicular diameter at the widest portion of the tumor and will be 
made and recorded by the physician or his designee.  An estimate of overall clinical objective 
response will be made and recorded at the end of treatment.   
 
9.2 Response Definitions (RECIST) 
9.2.1 Complete Pathological Response (PCR): Absence of all invasive tumor in both 
breast and axilla.   
9.2.2 Complete clinical response (CR). The patient must be free of all symptoms.  All 
measurable, evaluable and non-evaluable lesions and sites must be assessed and have 
resolved on physical exam and imaging.   
9.2.2  Partial Response (PR): Thirty percent or greater decrease in the sum of the products 
of two perpendicular diameters of all measured lesions.   No lesion may increase in size and 
no new lesions may appear 
9.2.3 Minor Response (MR): A decrease in measurable lesion(s) which is too small or too 
brief to qualify as a Partial Response.  MR is considered a treatment failure, but should be 
noted as a possible indicator of biologic activity. 
9.2.4 Progressive Disease (PD): Any increase of ≥25% in the sum of the products of 
diameters of any measurable lesion, in the reappearance of any lesion which had previously 
disappeared, or appearance of an unequivocal new lesion.   
 
9.2.5 Stable Disease (SD):  No change in tumor size(s) and no evidence of progression.  
Patients are classified with stable disease if at the first tumor assessment after the study 
treatment, there is neither disease progression nor a response that is later confirmed 
 
9.3 TIME TO PROGRESSION 
The primary endpoint of this study is time to progression. Documentation of first local or 
distant recurrence will be defined as progression of disease after enrollment. PFS will be 
measured from the enrollment. The patients will be followed clinically as outlined at 3, 6, and 
12 months with subsequent follow-up per standard of care guidelines as clinically indicated.    
 
 
10.0 CRITERIA FOR EVALUATION OF RESPONSE AND DISCONTINUATION OF THERAPY  
10.1 Response-evaluable: Participants will be considered response evaluable for immune study 
endpoints if they receive any aATC infusion.  All patients who begin neoadjuvant chemoT and 
complete planned surgery will be evaluable for pathologic responses. Those with complete 
pathologic responses are excluded from this study.   

V05/12/15 
31 
10.2 Disease Progression: Patients who progress during the neoadjuvant chemoT and/or  have 
residual disease after surgery are ineligible for this study. 
10.3 Extraordinary Medical Circumstances: If at any time the constraints of this protocol are 
detrimental to the patient’s health, the patient shall be withdrawn from treatment. Determination of 
whether the patient is evaluable for response will be determined by the criteria in 10.1 and 10.2. 
10.4 Patient decision to discontinue therapy 
10.5 Protocol violation: Unexplained delay in delivery of treatment. 
10.6 Grade 4 non-hematologic treatment related toxicity 
 
 
10.7 
Unexpected or Life-Threatening Toxicity: 
 
10.7.1 Questions regarding drug therapy will be directed to the Principal Investigator. 
 
10.7.2 Reporting requirements and procedures depend upon: (1) whether agents are sus-
pected of causing toxicity, (2) whether possibility of such a toxicity was presented in the 
consent form, or manufacturer’s literature (Published Toxicity), (3) the severity or grade of 
the toxicity. 
 
10.7.2.1 Expected toxicities: All expected toxicities related to leukapheresis 
and after first aATC infusion, including ongoing grade 3-4 hematologic toxicity 
related to chemoT, will be reported as part of regular data submission. 
 
10.7.2.2 Unexpected Toxicities: Unexpected toxicities are toxicities that are not 
listed in the toxicity management section of the protocol, the consent form, or 
the manufacturer’s package insert.  All unexpected toxicities related to 
leukapheresis and after first aATC infusion grade 3 or above will be reported 
per Wayne State University IRB guidelines. 
 
10.7.2.3 ChemoT, surgery, and radiation related toxicities: Since the chemoT, 
surgery, and radiation are standard treatments that will be managed by the 
treating (KCI or non-KCI) physicians and not overseen by the protocol, 
toxicities attributed to the chemoT, surgery, and radiation prior to first aATC 
infusion and toxicities related to surgery will not be collected or reported. 
 
10.7.2.4 All grade 5 toxicities occurring after registration and up to 30 days 
beyond the last aATC infusion will be reported to the Wayne State University 
IRB and the FDA per the standard guidelines of these organizations. 
 
 
11.0 REPORTING ADVERSE REACTIONS 
 
11.1 Investigators will notify the FDA and IRB of all serious and unexpected adverse drug 
reactions. 
 
11.2 All reactions in a 'reportable' category will be reported unless it is documented in the 
medical record chart that treatment is definitely not responsible for the toxicity. 
 
11.3 Serious and unexpected adverse reactions will be reported to the following sources 
within the stated time frame. 
 

V05/12/15 
32 
11.3.1 FDA: Written IND safety report within 7 calendar days.  The SAE will be 
reported by telephone within 3 working days.  SAE reports will be included in annual 
reports. 
 
11.3.2 All participating investigators: Written IND safety report within 7 calendar 
days. 
 
11.3.3 IRB: Written report within 10 working days. 
 
11.4  Procedure for calling the FDA is as follows: 
 
 
  
11.4.1 The research nurses or PI will call for ADRs. 
 
11.4.2 The PI or his designee will call for regulatory/protocol issues. 
 
11.4.3 The medical reviewer at the FDA to be identified: 
 
  
 
 
 
 
 
  
Food and Drug Administration 
 
  
Center for Biologics Evaluation and Research 
 
  
Document Control Center 
 
  
10903 New Hampshire Avenue 
 
  
Bldg. 71 G112 
 
  
Silver Spring, MD 20993-0002 
 
 
  
11.4.4  Document as completely as possible, and send copies to: 
 
  
Patient's chart 
 
  
FDA correspondence binder 
 
  
Protocol binder 
 
  
Lawrence G. Lum, M.D. 
 
  
 
12.0 Measurement of Functional Changes in Immune Populations: To measure functional 
changes in immune cell populations as a consequence of immunotherapy. Anti-tumor cytotoxicity 
will be examined.  Samples of serum will be stored for future evaluations such as cytokine and 
anti-tumor antibody immune responses.  The specific procedures are well-described in the 
literature.  Studies will be performed preIT (within 7 days or from leukaphresis product), pre-
infusion #5 (within 3 days), and 2 weeks (± 3 days) after IT.  Additional optional studies will be 
performed on specific tests if there are positive findings that warrant continued follow up for a 
particular patient at 2 weeks(± 3 days), 3 months (±1 week), 6 months (± 1 month), and 12 months 
(± 1 month) after IT.  If studies are still positive at 3 months, the optional studies can be obtained at 
6 month and 1 year (± 1 month).  The subsequent paragraphs summarize the strategy and 
procedures. 
 
 
12.1 Serum cytokine/chemokine levels.  Serum will be stored for subsequent cytokine 
analysis at the indicated time points.  
 
12.2 PBMC subsets and phenotype analysis.  Peripheral blood mononuclear cell (PBMC) 
and tumor-infiltrating lymphocyte (TIL) samples using immunohistochemistry to determine T 
cells and T cell subpopulations. 
  
12.3 Cytotoxic activity and IFNγ production in PBMC populations.  PBMC will be tested 
in specific cytotoxicity and IFNγ EliSpot assays after exposure to SK-BR-3 (HER2+ BrCa), or 
Daudi and/or Raji (Burkitt's lymphoma) at the indicated time points. Specific activity of pre-IT 
samples will be compared to those obtained during and post-IT as described in statistical 
analysis section.  

V05/12/15 
33 
 
13.0 Tumor Studies and Phenotypic Evaluations: 
 
13.1 Strategy: Standard IHC staining for ER, PR, and Her2/neu that will be performed by 
standard pathology procedures on the original diagnostic biopsy specimen by the Pathology 
Department at Wayne State University from the paraffin block.  Patients may be identified by 
their treating physicians. When a patient enrolls in this study, the formalin fixed sections from 
the patients will be retrieved and stained with anti-CD44, anti-CD24, and anti-CD133 (or 
other markers when they become available). The diagnostic sample will provide the baseline 
pre-chemoT result to be compared to formalin fixed sections from the surgical specimen. 
This strategy will optimize our ability to identify the changes after chemoT and before IT.  
 
 
13.2 Surgical Tumor Sample:  The tumor specimen will be divided for standard pathology 
and an aliquot for research to be stained for T cells and ALDH1. The paraffin block will be 
obtained from pathology where tumor was removed.  Sections from the block will be stained 
for T cell, T cell subsets, and tumor markers (see section 13.2). 
 
13.3 Immunohistochemistry Staining of Diagnostic and Surgical Specimens.  Standard 
IHC will be performed on formalin fixed tissue sections by Dr. Ali-Fehmi in pathology from the 
diagnostic, post chemoT, and surgical specimens.  These samples will provide an 
assessment of the changes in CD44, CD24, CD133, and other CSC markers (when 
available) that occur following chemoT and the subsequent effects of IT on the final surgical 
specimen.  ALDH1 will be the only marker not done on the diagnostic specimen. 
 
 14.0 STATISTICAL CONSIDERATIONS 
 
14.1 Objective: The primary objective is to determine  whether median PFSprovides 
sufficient evidence of efficacy to proceed with a larger study. . The secondary objective is to 
estimate functional and phenotypic changes from baseline in immune cell populations in 
blood and tumor samples, when accessible, as a function of IT, including  phenotype and 
anti-tumor functions (i.e. specific cytotoxicity and IFNγ EliSpots) of lymphocytes. The third 
objective is to investigate the association between proportion of infiltrating cells and cancer 
stem cells in the tumor at the time of surgery with time to progressive disease.. 
 
14.2 Design: This is a one-arm phase II study. 
 
14.3 Endpoints: The primary endpoint is  PFS, defined in 9.3. Secondary endpoints include 
immune responses measured before IT, once during IT and at 3 times after IT.  
 
14.4 Statistical Analyses:  
 
PFS will be estimated under the assumption of an exponential survival distribution. It will be 
concluded that median PFS exceeds 18 months if the estimated median PFS exceeds 25.2 
months. Kaplan-Meier methods will also be used to describe the PFS distribution  
. 
For those immune functions measured on a continuous scale the shape of the distribution will 
be assessed before analysis to be sure that the assumptions of the statistical procedures are 
not violated. If necessary either a data transformation will be used and parametric tests 
employed or rank tests will be used. If the assumptions are met with either the raw or 
transformed data, mixed effects models will be used to evaluate changes in continuous 
immune functions over time. We will assess the frequency distribution of immune functions 
measured on an ordinal scale and collapse categories if the data are too sparse. Changes in 
ordinal longitudinal measures will be assessed with generalized estimating equations using a 

V05/12/15 
34 
cumulative logistic link. We will also estimate the association between measures of immune 
function using rank correlation methods. Association between the amount of clinical disease , 
pCR or not, and the number of infiltrative cells in the tumor at the time of surgery and and 
immune function will be assessed using two-sample tests or two-sample. Association of PFS 
with immune function will be explored with univariable proportional hazards models. We will 
evaluate the simultaneous association of the immune function parameters with PFS in a 
multivariable model, expecting that the lack of independence between these measures will 
provide different results than the univariable models. 
 
Because of the relatively small sample size and the number of comparisons, correction for 
multiple comparisons will be made using Holm’s procedure. 
 
14.5 Statistical Power and Sample Size:  
Using the CRAB calculator (stattools.crab.org), sample size was calculated under the 
assumptions of an exponential distribution of PFS, a one-arm study, a one-sided test with 
type I error of 0.05 and statistical power of 80% to distinguish median PFS of 18 months from 
median PFS of 30 months.. It was further assumed that the accrual period will be 24 months 
and follow-up time 36 months. Under all of these assumptions, 32 evaluable patients are 
required. 
 
14.6 Accrual of Patients and Duration of Study: Patient accrual is expected to be 4 
patients/year so that the accrual period will last for 8 years. Interim analysis is not possible 
because it is a single group study and the accrual period is quite short. Immune follow-up 
studies will extend 1 year beyond the last HER2 bispecific antibody infusion based on positive 
results seen at the 1 month post-IT time-point.  
 
15.0 CLINICAL SAFETY MONITORING.   
The Data and Safety Monitoring Committee (DSMC) provides the primary oversight of data and safety 
monitoring at Karmanos Cancer Institute (KCI) for Investigator-initiated trials. 
 
15.1 
The Investigator will schedule monthly meetings or more frequently depending on the 
degree of risk encountered by study participants. These meetings will include the protocol 
investigators and research staff involved with the conduct of the protocol. 
15.2 
During these meetings the investigators review all aspects of the trial conduct related to: 
 
 Safety of protocol participants  
 Evaluate compliance with requirements regarding the reporting of AEs. 
 Validity and integrity of the data 
 Enrollment rate relative to expectation of target accrual 
 Retention of participants, protocol compliance (protocol violations and deviations) 
 Data completeness and source documentation 
 
These monthly meetings are recorded on the data and safety monitoring report forms and 
submitted for review on a quarterly basis to the DSMC.     
 
The Data Safety Monitoring Board (DSMB) of Karmanos Cancer Institute will perform 
comprehensive reviews of IND clinical trials on a semiannual basis. The Study 
Monitor must be familiar with the protocol, the investigational product, written 
informed consent, Standard Operating Procedures (SOPs), Good Clinical Practices 
(GCPs), and Applicable Regulatory Requirements.  
 
 
 
 

V05/12/15 
35 
 
16.0  REFERENCES.   
Reference List 
 
 1.  Guarneri V, Broglio K, Kau SW et al. Prognostic value of pathologic complete response after 
primary chemotherapy in relation to hormone receptor status and other factors. J.Clin.Oncol. 
2006;24:1037-1044. 
 2.  Carey LA, Dees EC, Sawyer L et al. The triple negative paradox: primary tumor 
chemosensitivity of breast cancer subtypes. Clin.Cancer Res. 2007;13:2329-2334. 
 3.  Liedtke C, Mazouni C, Hess KR et al. Response to neoadjuvant therapy and long-term survival 
in patients with triple-negative breast cancer. J.Clin.Oncol. 2008;26:1275-1281. 
 4.  Smigal C, Jemal A, Ward E et al. Trends in breast cancer by race and ethnicity: update 2006. 
CA Cancer J.Clin 2006;56:168-183. 
 5.  Carey LA, Perou CM, Livasy CA et al. Race, breast cancer subtypes, and survival in the 
Carolina Breast Cancer Study. JAMA 2006;295:2492-2502. 
 6.  Chlebowski RT, Chen Z, Anderson GL et al. Ethnicity and breast cancer: factors influencing 
differences in incidence and outcome. J.Natl.Cancer Inst. 2005;97:439-448. 
 7.  Kuerer HM, Newman LA, Smith TL et al. Clinical course of breast cancer patients with complete 
pathologic primary tumor and axillary lymph node response to doxorubicin-based neoadjuvant 
chemotherapy. J.Clin.Oncol. 1999;17:460-469. 
 8.  Abrial SC, Penault-Llorca F, Delva R et al. High prognostic significance of residual disease after 
neoadjuvant chemotherapy: a retrospective study in 710 patients with operable breast cancer. 
Breast Cancer Res.Treat. 2005;94:255-263. 
 9.  Wolmark N, Wang J, Mamounas E, Bryant J, Fisher B. Preoperative chemotherapy in patients 
with operable breast cancer: nine-year results from National Surgical Adjuvant Breast and Bowel 
Project B-18. J.Natl.Cancer Inst.Monogr 200196-102. 
 10.  Fisher B, Bryant J, Wolmark N et al. Effect of preoperative chemotherapy on the outcome of 
women with operable breast cancer. J.Clin.Oncol. 1998;16:2672-2685. 
 11.  Kaufmann M, Hortobagyi GN, Goldhirsch A et al. Recommendations from an international 
expert panel on the use of neoadjuvant (primary) systemic treatment of operable breast cancer: 
an update. J.Clin.Oncol. 2006;24:1940-1949. 
 12.  Kuroi K, Toi M, Tsuda H, Kurosumi M, Akiyama F. Unargued issues on the pathological 
assessment of response in primary systemic therapy for breast cancer. Biomed.Pharmacother. 
2005;59 Suppl 2:S387-S392. 
 13.  Bear HD, Anderson S, Smith RE et al. Sequential preoperative or postoperative docetaxel 
added to preoperative doxorubicin plus cyclophosphamide for operable breast cancer:National 
Surgical Adjuvant Breast and Bowel Project Protocol B-27. J.Clin.Oncol. 2006;24:2019-2027. 
 14.  Li X, Lewis MT, Huang J et al. Intrinsic resistance of tumorigenic breast cancer cells to 
chemotherapy. J.Natl.Cancer Inst. 2008;100:672-679. 
 15.  Charafe-Jauffret E, Ginestier C, Iovino F et al. Breast cancer cell lines contain functional cancer 
stem cells with metastatic capacity and a distinct molecular signature. Cancer Res 2009;69:1302-

V05/12/15 
36 
1313. 
 16.  Ginestier C, Hur MH, Charafe-Jauffret E et al. ALDH1 is a marker of normal and malignant 
human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 2007;1:555-
567. 
 17.  Croker AK, Goodale D, Chu J et al. High aldehyde dehydrogenase and expression of cancer 
stem cell markers selects for breast cancer cells with enhanced malignant and metastatic ability. 
J Cell Mol Med 2008 
 18.  Xiao Y, Ye Y, Yearsley K, Jones S, Barsky SH. The lymphovascular embolus of inflammatory 
breast cancer expresses a stem cell-like phenotype. Am J Pathol 2008;173:561-574. 
 19.  Wright MH, Calcagno AM, Salcido CD et al. Brca1 breast tumors contain distinct CD44+/. 
Breast Cancer Res 2008;10:R10. 
 20.  Disis ML, Cheever MA. Her-2/neu protein: a target for antigen-specific immunotherapy of 
human cancer. Adv.Cancer Res. 1997;71:343-371. 
 21.  Watanabe M, Nakada T, Yuta H. Analysis of protooncogene c-erbB-2 in benign and malignant 
human prostate. Int.Urol.Nephrol. 1999;31:61-73. 
 22.  Schwartz SJr, Caceres C, Morote J et al. Gains of the relative genomic content of erbB-1 and 
erbB-2 in prostate carcinoma and their association with metastasis. Int.J.Oncol. 1999;14:367-371. 
 23.  Morote J, de Torres I, Caceres C et al. Prognostic value of immunohistochemical expression of 
the c-erbB-2 oncoprotein in metastasic prostate cancer. Int.J.Cancer 1999;84:421-425. 
 24.  Molina MA, Codony-Servat J, Albanell J et al. Trastuzumab (herceptin), a humanized anti-Her2 
receptor monoclonal antibody, inhibits basal and activated Her2 ectodomain cleavage in breast 
cancer cells Cancer Res. 2001;61:4744-4749. 
 
 25.  Le XF, Claret FX, Lammayot A et al. The role of cyclin-dependent kinase inhibitor p27Kip1 in 
anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition. J Biol.Chem. 
2003;278:23441-23450. 
 
 26.  Jackson JG, St Clair P, Sliwkowski MX, Brattain MG. Blockade of epidermal growth factor- or 
heregulin-dependent ErbB2 activation with the anti-ErbB2 monoclonal antibody 2C4 has 
divergent downstream signaling and growth effects Cancer Res. 2004;64:2601-2609. 
 
 27.  Yakes 
FM, 
Chinratanalab 
W, 
Ritter 
CA 
et 
al. 
Herceptin-induced 
inhibition 
of 
phosphatidylinositol-3 kinase and Akt Is required for antibody-mediated effects on p27, cyclin D1, 
and antitumor action. Cancer Res. 2002;62:4132-4141. 
 
 28.  Mohsin SK, Weiss HL, Gutierrez MC et al. Neoadjuvant trastuzumab induces apoptosis in 
primary breast cancers J Clin.Oncol. 2005;23:2460-2468. 
 
 29.  Cooley S, Burns LJ, Repka T, Miller JS. Natural killer cell cytotoxicity of breast cancer targets is 
enhanced by two distinct mechanisms of antibody-dependent cellular cytotoxicity against LFA-3 
and HER2/neu Exp.Hematol. 1999;27:1533-1541. 
 
 30.  Clynes RA, Towers TL, Presta LG, Ravetch JV. Inhibitory Fc receptors modulate in vivo 
cytoxicity against tumor targetsNat.Med. 2000;6:443-446. 
 
 31.  Carson WE, Parihar R, Lindemann MJ et al. Interleukin-2 enhances the natural killer cell 

V05/12/15 
37 
response to Herceptin-coated Her2/neu-positive breast cancer cells. Eur.J Immunol. 
2001;31:3016-3025. 
 
 32.  Repka T, Chiorean EG, Gay J et al. Trastuzumab and interleukin-2 in HER2-positive metastatic 
breast cancer: a pilot study Clin.Cancer Res. 2003;9:2440-2446. 
 
 33.  Gennari R, Menard S, Fagnoni F et al. Pilot study of the mechanism of action of preoperative 
trastuzumab in patients with primary operable breast tumors overexpressing HER2 Clin.Cancer 
Res. 2004;10:5650-5655. 
 
 34.  Cobleigh MA, Vogel CL, Tripathy D et al. Multinational study of the efficacy and safety of 
humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastatic 
breast cancer that has progressed after chemotherapy for metastatic disease J Clin.Oncol. 
1999;17:2639-2648. 
 
 35.  Vogel CL, Cobleigh MA, Tripathy D et al. Efficacy and safety of trastuzumab as a single agent 
in first-line treatment of HER2-overexpressing metastatic breast cancer J Clin.Oncol. 
2002;20:719-726. 
 
 36.  Pegram M, Hsu S, Lewis G et al. Inhibitory effects of combinations of HER-2/neu antibody and 
chemotherapeutic agents used for treatment of human breast cancers Oncogene 1999;18:2241-
2251. 
 
 37.  Slamon DJ, Clark GM, Wong SG et al. Human breast cancer: correlation of relapse and survival 
with amplification of the HER-2/neu oncogene. Science 1987;235:177-182. 
 38.  Cuello M, Ettenberg SA, Clark AS et al. Down-regulation of the erbB-2 receptor by trastuzumab 
(herceptin) enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis 
in breast and ovarian cancer cell lines that overexpress erbB-2. Cancer Res. 2001;61:4892-4900. 
 39.  Lum LG, Rathore R, Cummings F et al. Phase I/II study of treatment of stage IV breast cancer 
with OKT3 x trastuzumab-armed activated T cells. Clin.Breast Cancer 2003;4:212-217. 
 40.  Davol PA, Lum LG. How important is HER2/neu amplification and expression when selecting 
patients for HER2/neu-targeted therapies? Clin.Breast Cancer 2004;5:70-71. 
 41.  Smith IE, A'Hern RP, Coombes GA et al. A novel continuous infusional 5-fluorouracil-based 
chemotherapy regimen compared with conventional chemotherapy in the neo-adjuvant treatment 
of early breast cancer: 5 year results of the TOPIC trial. Ann.Oncol. 2004;15:751-758. 
 42.  Chua S, Smith IE, A'Hern RP et al. Neoadjuvant vinorelbine/epirubicin (VE) versus standard 
adriamycin/cyclophosphamide (AC) in operable breast cancer: analysis of response and 
tolerability in a randomised phase III trial (TOPIC 2). Ann.Oncol. 2005;16:1435-1441. 
 43.  Buzdar AU, Singletary SE, Theriault RL et al. Prospective evaluation of paclitaxel versus 
combination chemotherapy with fluorouracil, doxorubicin, and cyclophosphamide as neoadjuvant 
therapy in patients with operable breast cancer. J.Clin.Oncol. 1999;17:3412-3417. 
 44.  Guarneri V, Frassoldati A, Giovannelli S, Borghi F, Conte P. Primary systemic therapy for 
operable breast cancer: a review of clinical trials and perspectives. Cancer Lett. 2007;248:175-
185. 
 45.  de MA, Nuzzo F, D'Aiuto G et al. Docetaxel plus epidoxorubicin as neoadjuvant treatment in 
patients with large operable or locally advanced carcinoma of the breast: a single-center, phase II 
study. Cancer 2002;94:895-901. 

V05/12/15 
38 
 46.  Malhotra V, Dorr VJ, Lyss AP et al. Neoadjuvant and adjuvant chemotherapy with doxorubicin 
and docetaxel in locally advanced breast cancer. Clin.Breast Cancer 2004;5:377-384. 
 47.  Espinosa E, Morales S, Borrega P et al. Docetaxel and high-dose epirubicin as neoadjuvant 
chemotherapy in locally advanced breast cancer. Cancer Chemother.Pharmacol. 2004;54:546-
552. 
 48.  Wenzel C, Locker GJ, Schmidinger M et al. Combined analysis of two phase II trials in patients 
with primary and advanced breast cancer with epidoxorubicin and docetaxel+granulocyte colony 
stimulating factor. Anticancer Drugs 2002;13:67-74. 
 49.  Ganem G, Tubiana-Hulin M, Fumoleau P et al. Phase II trial combining docetaxel and 
doxorubicin as neoadjuvant chemotherapy in patients with operable breast cancer. Ann.Oncol. 
2003;14:1623-1628. 
 50.  Gogas H, Papadimitriou C, Kalofonos HP et al. Neoadjuvant chemotherapy with a combination 
of pegylated liposomal doxorubicin (Caelyx) and paclitaxel in locally advanced breast cancer: a 
phase II study by the Hellenic Cooperative Oncology Group. Ann.Oncol. 2002;13:1737-1742. 
 51.  Moliterni A, Tarenzi E, Capri G et al. Pilot study of primary chemotherapy with doxorubicin plus 
paclitaxel in women with locally advanced or operable breast cancer. Semin.Oncol. 1997;24:S17. 
 52.  Bellino R, Cortese P, Danese S et al. Epidoxorubicin and paclitaxel as primary chemotherapy 
for T > 3 cm and T4 breast cancer patients. Anticancer Res. 2000;20:4825-4828. 
 53.  Schneeweiss A, Huober J, Sinn HP et al. Gemcitabine, epirubicin and docetaxel as primary 
systemic therapy in patients with early breast cancer: results of a multicentre phase I/II study. 
Eur.J.Cancer 2004;40:2432-2438. 
 54.  Conte PF, Donati S, Gennari A et al. Primary chemotherapy with gemcitabine, epirubicin and 
taxol (GET) in operable breast cancer: a phase II study. Br.J.Cancer 2005;93:406-411. 
 55.  Evans TR, Yellowlees A, Foster E et al. Phase III randomized trial of doxorubicin and docetaxel 
versus doxorubicin and cyclophosphamide as primary medical therapy in women with breast 
cancer: an anglo-celtic cooperative oncology group study. J.Clin.Oncol. 2005;23:2988-2995. 
 56.  Dieras V, Fumoleau P, Romieu G et al. Randomized parallel study of doxorubicin plus paclitaxel 
and doxorubicin plus cyclophosphamide as neoadjuvant treatment of patients with breast cancer. 
J.Clin.Oncol. 2004;22:4958-4965. 
 57.  Steger GG, Galid A, Gnant M et al. Pathologic complete response with six compared with three 
cycles of neoadjuvant epirubicin plus docetaxel and granulocyte colony-stimulating factor in 
operable breast cancer: results of ABCSG-14. J.Clin.Oncol. 2007;25:2012-2018. 
 58.  Reitsamer R, Peintinger F, Prokop E, Hitzl W. Pathological complete response rates comparing 
3 versus 6 cycles of epidoxorubicin and docetaxel in the neoadjuvant setting of patients with 
stage II and III breast cancer. Anticancer Drugs 2005;16:867-870. 
 59.  Smith IC, Heys SD, Hutcheon AW et al. Neoadjuvant chemotherapy in breast cancer: 
significantly enhanced response with docetaxel. J.Clin.Oncol. 2002;20:1456-1466. 
 60.  Heys SD, Hutcheon AW, Sarkar TK et al. Neoadjuvant docetaxel in breast cancer: 3-year 
survival results from the Aberdeen trial. Clin.Breast Cancer 2002;3 Suppl 2:S69-S74. 
 61.  von MG, Blohmer JU, Raab G et al. In vivo chemosensitivity-adapted preoperative 

V05/12/15 
39 
chemotherapy in patients with early-stage breast cancer: the GEPARTRIO pilot study. Ann.Oncol. 
2005;16:56-63. 
 62.  Green MC, Buzdar AU, Smith T et al. Weekly paclitaxel improves pathologic complete 
remission in operable breast cancer when compared with paclitaxel once every 3 weeks. 
J.Clin.Oncol. 2005;23:5983-5992. 
 63.  Baldini E, Gardin G, Giannessi PG et al. Accelerated versus standard cyclophosphamide, 
epirubicin and 5-fluorouracil or cyclophosphamide, methotrexate and 5-fluorouracil: a randomized 
phase III trial in locally advanced breast cancer. Ann.Oncol. 2003;14:227-232. 
 64.  Miller KD, Caskill-Stevens W, Sisk J et al. Combination versus sequential doxorubicin and 
docetaxel as primary chemotherapy for breast cancer: A randomized pilot trial of the Hoosier 
Oncology Group. J.Clin.Oncol. 1999;17:3033-3037. 
 65.  von MG, Costa SD, Eiermann W et al. Maximized reduction of primary breast tumor size using 
preoperative chemotherapy with doxorubicin and docetaxel. J.Clin.Oncol. 1999;17:1999-2005. 
 66.  Cooper BW, Radivoyevitch T, Overmoyer BA et al. Phase II study of dose-dense sequential 
doxorubicin and docetaxel for patients with advanced operable and inoperable breast cancer. 
Breast Cancer Res.Treat. 2006;97:311-318. 
 67.  von MG, Raab G, Caputo A et al. Doxorubicin with cyclophosphamide followed by docetaxel 
every 21 days compared with doxorubicin and docetaxel every 14 days as preoperative treatment 
in operable breast cancer: the GEPARDUO study of the German Breast Group. J.Clin.Oncol. 
2005;23:2676-2685. 
 68.  Perou CM, Sorlie T, Eisen MB et al. Molecular portraits of human breast tumours. Nature 
2000;406:747-752. 
 69.  Van d, V, He YD, van't Veer LJ et al. A gene-expression signature as a predictor of survival in 
breast cancer. N.Engl.J.Med. 2002;347:1999-2009. 
 70.  Ayers M, Symmans WF, Stec J et al. Gene expression profiles predict complete pathologic 
response to neoadjuvant paclitaxel and fluorouracil, doxorubicin, and cyclophosphamide 
chemotherapy in breast cancer. J.Clin.Oncol. 2004;22:2284-2293. 
 71.  Chang JC, Wooten EC, Tsimelzon A et al. Gene expression profiling for the prediction of 
therapeutic response to docetaxel in patients with breast cancer. Lancet 2003;362:362-369. 
 72.  Chang JC, Wooten EC, Tsimelzon A et al. Patterns of resistance and incomplete response to 
docetaxel by gene expression profiling in breast cancer patients. J.Clin.Oncol. 2005;23:1169-
1177. 
 73.  Rouzier R, Perou CM, Symmans WF et al. Breast cancer molecular subtypes respond 
differently to preoperative chemotherapy. Clin.Cancer Res. 2005;11:5678-5685. 
 74.  Thuerigen O, Schneeweiss A, Toedt G et al. Gene expression signature predicting pathologic 
complete response with gemcitabine, epirubicin, and docetaxel in primary breast cancer. 
J.Clin.Oncol. 2006;24:1839-1845. 
 75.  Gianni L, Zambetti M, Clark K et al. Gene expression profiles in paraffin-embedded core biopsy 
tissue predict response to chemotherapy in women with locally advanced breast cancer. 
J.Clin.Oncol. 2005;23:7265-7277. 

V05/12/15 
40 
 76.  Lizee G, Radvanyi LG, Overwijk WW, Hwu P. Improving antitumor immune responses by 
circumventing immunoregulatory cells and mechanisms. Clin.Cancer Res. 2006;12:4794-4803. 
 77.  Wang LX, Li R, Yang G et al. Interleukin-7-dependent expansion and persistence of melanoma-
specific T cells in lymphodepleted mice lead to tumor regression and editing. Cancer Res. 
2005;65:10569-10577. 
 78.  Gattinoni L, Finkelstein SE, Klebanoff CA et al. Removal of homeostatic cytokine sinks by 
lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8+ T cells. 
J.Exp.Med. 2005;202:907-912. 
 79.  Wang LX, Shu S, Plautz GE. Host lymphodepletion augments T cell adoptive immunotherapy 
through enhanced intratumoral proliferation of effector cells. Cancer Res. 2005;65:9547-9554. 
 80.  Dummer W, Niethammer AG, Baccala R et al. T cell homeostatic proliferation elicits effective 
antitumor autoimmunity. J.Clin.Invest 2002;110:185-192. 
 81.  Cho BK, Rao VP, Ge Q, Eisen HN, Chen J. Homeostasis-stimulated proliferation drives naive T 
cells to differentiate directly into memory T cells. J.Exp.Med. 2000;192:549-556. 
 82.  Nobusada K, Kuhn L, Pilon-Thomas SA, Mule JJ. Adoptive Immunotherapy Combined With A 
Dendritic Cell-Based Vaccine Results in the Rejection of Established Tumor in A Murine 
Melanoma Model [abstract]. J.Immunother. 2007;28:614. 
 83.  Ma J, Urba WJ, Si L et al. Anti-tumor T cell response and protective immunity in mice that 
received sublethal irradiation and immune reconstitution. Eur.J.Immunol. 2003;33:2123-2132. 
 84.  Hu HM, Poehlein CH, Urba WJ, Fox BA. Development of antitumor immune responses in 
reconstituted lymphopenic hosts. Cancer Res. 2002;62:3914-3919. 
 85.  Dudley ME, Wunderlich JR, Yang JC et al. Adoptive cell transfer therapy following non-
myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory 
metastatic melanoma. J.Clin.Oncol. 2005;23:2346-2357. 
 86.  Mullins DW, Burger CJ, Elgert KD. Paclitaxel enhances macrophage IL-12 production in tumor-
bearing hosts through nitric oxide. J.Immunol. 1999;162:6811-6818. 
 87.  Kubo M, Morisaki T, Matsumoto K et al. Paclitaxel probably enhances cytotoxicity of natural 
killer cells against breast carcinoma cells by increasing perforin production. Cancer 
Immunol.Immunother. 2005;54:468-476. 
 88.  Zhang L, Dermawan K, Jin M et al. Differential impairment of regulatory T cells rather than 
effector T cells by paclitaxel-based chemotherapy. Clin Immunol. 2008 
 89.  Machiels JP, Reilly RT, Emens LA et al. Cyclophosphamide, doxorubicin, and paclitaxel 
enhance the antitumor immune response of granulocyte/macrophage-colony stimulating factor-
secreting whole-cell vaccines in HER-2/neu tolerized mice. Cancer Res. 2001;61:3689-3697. 
 90.  Weiner LM, Clark JI, Davey M et al. Phase I trial of 2B1, a bispecific monoclonal antibody 
targeting c-erbB-2 and Fc gamma RIII. Cancer Res. 1995;55:4586-4593. 
 91.  Valone FH, Kaufman PA, Guyre PM et al. Phase Ia/Ib trial of bispecific antibody MDX-210 in 
patients with advanced breast or ovarian cancer that overexpresses the proto-oncogene HER-
2/neu. J.Clin.Oncol. 1995;13:2281-2292. 

V05/12/15 
41 
 92.  Hayden MS, Linsley PS, Gayle MA et al. Single-chain mono- and bispecific antibody derivates 
with novel biological properties and antitumor activity from a COS cell transient expression 
system. Therapeut.Immunol. 1994;1:3-15. 
 93.  Shalaby MR, Shepard HM, Presta L et al. Development of humanized bispecific antibodies 
reactive with cytotoxic lymphocytes and tumor cells overexpressing the HER2 protooncogene. 
J.Exp.Med. 1992;175:217-225. 
 94.  Zhu Z, Lewis GD, Carter P. Engineering high affinity humanized anti-p185HER2/anti-CD3 
bispecific F(ab')2 for efficient lysis of p185HER2 overexpressing tumor cells. Int.J.Cancer 
1995;62:319-324. 
 95.  Kuwahara M, Kuroki M, Arakawa F et al. A mouse/human-chimeric bispecific antibody reactive 
with human carcinoembryonic antigen-expressing cells and human T-lymphocytes. Anticancer 
Res. 1997;16:2661-2668. 
 96.  Nakamura Y, Tokuda Y, Iwasawa M et al. Large-scale culture system of human CD4+ 
helper/killer T cells for the application of adoptive tumour immunotherapy [Comment in: 
Br.J.Cancer 1993; 67:865-7]. Br.J.Cancer 1992;66:20-26. 
 97.  James ND, Atherton PJ, Jones AJ et al. A phase II study of the bispecific antibody MDX-H210 
(anti-HER2 x CD 64) with GM-CSF in Her2+ advanced prostrate cancer. Br.J.Cancer 
2001;85:152-156. 
 98.  Ochoa AC, Gromo G, Alter BJ, Sondel PM, Bach FH. Long-term growth of lymphokine-activated 
killer (LAK) cell: role of anti-CD3, beta-IL 1, interferon-gamma and -beta. J.Immunol. 
1987;138:2728-2733. 
 99.  Anderson PM, Bach FH, Ochoa AC. Augmentation of cell number and LAK activity in peripheral 
blood mononuclear cells activated with anti-CD3 and interleukin-2. Preliminary results in children 
with acute lymphocytic leukemia and neuroblastoma. Cancer Immunol.Immunother. 1988;27:82-
88. 
100.  Chen BP, Malkovsky M, Hank JA, Sondel PM. Nonrestricted cytotoxicity mediated by interleukin 
2-expanded leukocytes is inhibited by anti-LFA-1 monoclonal antibodies (MoAb) but potentiated 
by anti-CD3 MoAB. Cell Immunol. 1987;110:282-293. 
101.  Lotzova E, Savary CA, Herberman RB et al. Augmentation of antileukemia lytic activity by OKT3 
monoclonal antibody: synergism of OKT3 and interleukin-2. Nat.Immun.Cell Growth Regul. 
1987;6:219-223. 
102.  Yang SC, Fry KD, Grimm EA, Roth JA. Successful combination immunotherapy for the 
generation in vivo of antitumor activity with anti-CD3, interleukin 2, and tumor necrosis factor 
alpha. Arch.Surg. 1990;125:220-225. 
103.  Ueda M, Joshi ID, Dan M et al. Preclinical studies for adoptive immunotherapy in bone marrow 
transplantation:  II.  Generation of anti-CD3 activated cytotoxic T cells from normal donors and 
autologous bone marrow transplant candidates. Transplantation 1993;56:351-356. 
104.  Uberti JP, Joshi I, Ueda M et al. Preclinical studies using immobilized OKT3 to activate human 
T cells for adoptive immunotherapy: optimal conditions for the proliferation and induction of non-
MHC restricted cytotoxicity. Clin.Immunol.Immunopathol. 1994;70:234-240. 
105.  Anderson PM, Blazar BR, Bach FH, Ochoa AC. Anti-CD3 + IL-2-stimulated murine killer cells: in 
vitro generation and in vivo antitumor activity. J.Immunol. 1989;142:1383-1394. 

V05/12/15 
42 
106.  Anderson PM, Ochoa AC, Ramsay NKC, Hasz D, Weisdorf D. Anti-CD3 + interleukin-2 
stimulation of marrow and blood: comparison of proliferation and cytotoxicity. Blood 
1992;80:1846-8153. 
107.  Ting C-C, Hargrove ME, Yun YS. Augmentation by anti-T3 antibody of the lymphokine-activated 
killer cell-mediated cytotoxicity. J.Immunol. 1988;141:741-748. 
108.  Ochoa AC, Hasz DE, Rezonzew R, Anderson PM, Bach FH. Lymphokine-activated killer activity 
in long-term cultures with anti-CD3 plus interleukin 2: identification and isolation of effector 
subsets. Cancer Res. 1989;49:963-968. 
109.  Van Wauwe JP, De Mey JR, Gooseens JG. OKT3: a monoclonal anti-human T lymphocyte 
antibody with potent mitogenic properties. J.Immunol. 1980;124:2708-2713. 
110.  Meuer SC, Hodgdon JC, Hussey RE et al. Antigen-like effects of monoclonal antibodies 
directed at receptors on human T cell clones. J.Exp.Med. 1983;158:988-993. 
111.  Meuer SC, Hussey RE, Cantrell DA et al. Triggering of the T3-Ti anti-receptor complex results 
in clonal T cell proliferation through an interleukin 2 dependent autocrine pathway. 
Proc.Natl.Acad.Sci.USA 1984;81:1509-1513. 
112.  Weiss A, Imboden JB. Cell surface molecules and early events involved in human T lymphocyte 
activation. Adv.Immunol. 1987;41:1-38. 
113.  Heath WR, Belz GT, Behrens GM et al. Cross-presentation, dendritic cell subsets, and the 
generation of immunity to cellular antigens. Immunol.Rev. 2004;199:9-26. 
114.  Lake RA, Robinson BW. Immunotherapy and chemotherapy--a practical partnership. 
Nat.Rev.Cancer 2005;5:397-405. 
115.  Nowak AK, Lake RA, Marzo AL et al. Induction of tumor cell apoptosis in vivo increases tumor 
antigen cross-presentation, cross-priming rather than cross-tolerizing host tumor-specific CD8 T 
cells. J.Immunol. 2003;170:4905-4913. 
116.  Sen M, Wankowski DM, Garlie NK et al. Use of anti-CD3 x anti-HER2/neu bispecific antibody 
for redirecting cytotoxicity of activated T cells toward HER2/neu Tumors. Journal of 
Hematotherapy & Stem Cell Research 2001;10:247-260. 
117.  Lum LG, Davol P, Grabert R et al. Targeting Pancreatic Cancer with Armed Activated T Cells 
Directed at Her2/neu Receptors. [abstract]. Exp.Hematol. 2002; 
118.  Davol PA, Smith JA, Kouttab N, Elfenbein GJ, Lum LG. Anti-CD3 x Anti-HER2 bispecific 
antibody effectively redirects armed T cells to inhibit tumor development and growth in hormone-
refractory prostate cancer-bearing SCID-Beige mice. Clin.Prostate Cancer 2004;3:112-121. 
119.  Irvine DJ, Purbhoo MA, Krogsgaard M, Davis MM. Direct observation of ligand recognition by T 
cells. Nature 2002;419:845-849. 
120.  Grabert RC, Cousens LP, Smith JA et al. Human T cells armed with Her2/neu bispecific 
antibodies divide, are cytotoxic, and secrete cytokines with repeated stimulation. Clin.Cancer 
Res. 2006;12:569-576.